Ctla-4 aptamer conjugates

ABSTRACT

Provided herein are, inter alfa, nucleic acid compounds useful for targeting CTLA-4-expressing cells and modulating cell activity of the CTLA-4-expressing cells. The compositions provided herein may be part of pharamceutcial compositions and may be used for treatment of cancer, inflammatory diseases, infectious diseases or metabolic diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/602,182, filed Jan. 21, 2015, which claims the benefit of U.S.Provisional Application No. 61/929,832, filed Jan. 21, 2014, which ishereby incorporated in its entirety and for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under grant numbersR01CA122976, R01CA146092, P50CA107399, and P30CA033572 awarded by theNational Institutes of Health. The Government has certain rights in theinvention.

REFERENCE TO A SEQUENCE LISTING, A TABLE OR A COMPUTER PROGRAM LISTINGAPPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file048440-538C01US_SEQUENCE_LISTING_ST25.TXT, created on Jun. 3, 2020,2,181 bytes, machine format IBM-PC, MS Windows operating system, ishereby incorporated by reference

BACKGROUND OF THE INVENTION

Recent promising human results of immunotherapies to block immunecheckpoints such as cytotoxic T-lymphocyte-associated antigen 4 (CTLA4)and programmed cell death protein 1 (PD-1) (Pardoll, D. M., Nat RevCancer 12:252-264 (2012); Pardoll, D. M., Nat Immunol 13:1129-1132(2012); Keir, M. E. et al., Annu Rev Immunol 26:677-704 (2008))illustrate the importance of targeting molecules that inhibit Tcell-mediated antitumor immunity. However, the immunosuppressive tumormicroenvironment hampers the success of various immunotherapies. Thereare several intracellular checkpoints with great potential as targets topromote potent antitumor immunity. STAT3, for example, has been shown tobe a crucial signaling mediator in tumor-associated immune cells, aswell as in tumor cells (Yu, H. et al., Nat Rev Cancer 9:798-809 (2009);Kortylewski, M. and Yu, H., Curr Opin Immunol 20:228-233 (2008);Kortylewski, M. et al., Nat Med 11:1314-1321 (2005); Herrmann, A. etal., Cancer Res 70:7455-7464 (2010)). In tumor cells, STAT3 promotestumor cell survival/proliferation, invasion, and immunosuppression (Yu,H., and Jove, R., Nat Rev Cancer 4:97-105 (2004)). In the tumormicroenvironment, STAT3 is persistently activated in immune cells,including T cells (Kujawski, M. et al., Cancer Res 70:9599-9610 (2010);Yu, H. et al., Nat Rev Immunol 7:41-51 (2007)). CD4⁺ T regulatory cells(T_(Regs)) can induce peripheral tolerance, suppressing CD8 T cellfunctions in various diseases including cancer (Kortylewski, M. et al.,Nat Med 11:1314-1321 (2005); Curiel, T. J. et al., Nat Med 10:942-949(2004); Shevach, E. M., Nat Rev Immunol 2:389-400 (2002); Chen, M. L. etal., Proc Natl Acad Sci USA 102:419-424 (2005); Mempel, T. R. et al.,Immunity 25:129-141 (2006); Arens, R. and Schoenberger, S. P., ImmunolRev 235:190-205 (2010)). Activated STAT3 in T cells contributes toexpanding tumor-associated CD4⁺ T_(Regs) (Kortylewski, M. et al., NatMed 11:1314-1321 (2005); Pallandre, J. R. et al., J Immunol179:7593-7604 (2007)). Moreover, Stat3^(−/−) CD8⁺ T cells, bothendogenous or adoptively transferred, mount potent antitumor immuneresponses compared to those with intact Stat3 (Kujawski, M. et al.,Cancer Res 70:9599-9610 (2010)).

As a nuclear transcription factor lacking its own enzymatic activity,STAT3 is a challenging target for both antibody and small-molecule drugs(Yu, H., and Jove, R., Nat Rev Cancer 4:97-105 (2004); Darnell, J. E.,Nat Med 11:595-596 (2005); Darnell, J. E., Jr., Nat Rev Cancer 2:740-749(2002)). Recent pioneering work has shown the feasibility to deliversiRNA into tumor cells in vivo (McNamara, J. O., 2nd et al., NatBiotechnol 24:1005-1015 (2006)). In particular, chimeric RNAs orDNA-RNAs consisting of a siRNA fused to nucleic acid sequences, whichbind to either a cell surface ligand or an intracellular receptor withhigh affinity, have demonstrated therapeutic efficacy in preclinicalmodels (McNamara, J. O., 2nd et al., Nat Biotechnol 24:1005-1015 (2006);Wheeler, L. A. et al., J Clin Invest 121:2401-2412 (2011); Kortylewski,M. et al., Nat Biotechnol 27:925-932 (2009)). The majority of such siRNAdelivery technologies involves the fusion of siRNA to an aptamer, astructured RNA with high affinity to epitopes on tumor cells and virallyinfected epithelial cells. Applicants recently described a technologyfor efficient in vivo delivery of siRNA into immune cells by linking ansiRNA to CpG oligonucleotide, which binds to its cognate receptor, TLR9(Kortylewski, M. et al., Nat Biotechnol 27:925-932 (2009)). TLR9 isexpressed intracellularly in cells of myeloid lineage and B cells, aswell as tumor cells expressing TLR9, including human leukemic cells(Kortylewski, M. et al., Nat Biotechnol 27:925-932 (2009); Zhang, Q. etal., Blood 121:1304-1315 (2013)). However, the CpG-siRNA approach doesnot directly target T cells (Kortylewski, M. et al., Nat Biotechnol27:925-932 (2009)).

Recently, an effective way to deliver siRNA into CD4 T cells for localtreatment of HIV has been developed (Wheeler, L. A. et al., J ClinInvest 121:2401-2412 (2011)). However, for cancer immunotherapy, it isalso crucial to regulate CD8⁺ effector T cells, in addition to CD4⁺cells. Further, it is quite plausible that selectively targeting thesubpopulations of CD8⁺ and CD4⁺ T cells in the tumor microenvironment,rather than T cells in general, should afford more antitumor efficacywhile reducing toxicity. The expression of CTLA4 is dysregulated intumors and in tumor-associated T cells and is a promisingimmunotherapeutic target (Santulli-Marotto, S. et al., Cancer Res63:7483-7489 (2003)). The broad antitumor immune response by CTLA4blockade is thought to be mainly mediated by CD4⁺ T cells: reducingT_(Regs) and increasing helper T cells (Chen, M. L. et al., Proc NatlAcad Sci USA 102:419-424 (2005); Wing, K. et al., Science 322:271-275(2008); Byrne, W. L. et al., Cancer Res 71:6915-6920 (2011); Peggs, K.S. et al., J Exp Med 206:1717-1725 (2009); Lenschow, D. J. et al., AnnuRev Immunol 14:233-258 (1996)). However, activated/exhausted CD8 T cellsalso express CTLA4 (Walunas, T. L. et al., Immunity 1:405-413 (1994);Teft, W. A. et al., Annu Rev Immunol 24:65-97 (2006); Wherry, E. J. etal., Immunity 27:670-684 (2007)).

There is a need in the art for compositions and methods of deliveringmodulators of cell activity (e.g., anti-tumor agents, anti-obesityagents) to cells (e.g., malignant cells, tumor-associated T cells,effector T cells) to inhibit diseases such as cancer, metastasis ormetabolic diseases. The nucleic acid compounds and methods of using thesame as provided herein cure these and other needs in the art.

BRIEF SUMMARY OF THE INVENTION

Provided herein are, inter alia, nucleic acid compounds useful fortargeting CTLA-4-expressing cells and modulating cell activity of theCTLA-4-expressing cells. The compositions provided herein may be part ofpharamceutcial compositions and may be used for treatment of a varietyof different disease. For example, the compositions provided herein maybe used in the treatment of cancer, inflammatory diseases, infectiousdiseases and metabolic diseases (e.g., diabetes).

In one aspect a nucleic acid compound including a monomeric CTLA-4aptamer nucleic acid conjugated to a cell activity modulating nucleicacid is provided.

In another aspect, a mammalian cell including a nucleic acid compound asprovided herein including embodiments thereof is provided.

In another aspect, a cellular receptor bound to a nucleic acid compoundas provided herein including embodiments thereof is provided.

In another aspect, a pharmaceutical composition including apharmaceutically acceptable excipient and a nucleic acid compound asprovided herein including embodiments thereof is provided.

In another aspect, a method of treating a disease in a subject in needthereof is provided. The method includes administering to a subject atherapeutically effective amount of a nucleic acid compound as providedherein including embodiments thereof, thereby treating the subject.

In another aspect, a method of inhibiting growth of a cancer cell isprovided. The method includes contacting a cancer cell with a nucleicacid compound as provided herein including embodiments thereof, therebyinhibiting growth of the cancer cell.

In another aspect, a method of inhibiting activity of a tumor-associatedT cell is provided. The method includes contacting a tumor-associated Tcell with a nucleic acid compound as provided herein includingembodiments thereof, thereby inhibiting activity of the tumor-associatedT cell.

In another aspect, a method of stimulating the immune system of asubject in need thereof is provided. The method includes administeringto the subject an effective amount of a nucleic acid compound asprovided herein including embodiments thereof to a subject, therebystimulating the immune system of the subject.

In another aspect, a method of inhibiting a protein activity in a cellis provided. The method includes contacting a cell with a nucleic acidcompound as provided herein including embodiments thereof, therebyforming a contacted cell. The contacted cell is allowed to express theantisense nucleic acid thereby forming a cellular antisense nucleicacid. The cellular antisense nucleic acid is allowed to inhibit aprotein activity in the cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1H: CTLA4^(apt)-siRNA uptake and gene silencing in T cellsincluding CD8⁺ T cells. (FIG. 1A) CTLA4^(apt)-siRNA^(FITc) positive andnegative CD3⁺ T cells (CD3⁺ FITC⁺ and CD3⁺FITC⁻) were isolated by FACSsorting from tumors of mice treated as indicated. Expression of Stat3mRNA was assessed by RT-PCR. SD shown. Two-tailed student's t-test *)P<0.05, **) P<0.01, ***) P<0.001. (FIG. 1B) Cellular internalization ofCTLA-4^(apt)Stat3siRNA into a CD8⁺ T cell suspension was visualized byconfocal microscopy. Scale bar 5 μm. (FIG. 1C) Efficient uptake offluorescently labeled CTLA-4^(apt.)Stat3siRNA by CD8⁺ T cells. Flowcytometry analysis showing CD8⁺ T cells positive forCTLA-4^(apt.)Stat3siRNA after 2 h of treatment. (FIG. 1D) Intracellulartrafficking of CTLA-4^(apt.)Stat3siRNA through the endosomal compartmentindicated by EEA-1 staining was assessed by confocal analysis of CD8 Tcells treated for time points as indicated. Scale bar 5 μm. (FIG. 1E)Stat3 knock-down efficiency in vitro in CD8⁺ T cells byCTLA-4^(apt.)Stat3siRNA. Stat3 expression was analyzed by RT-PCR at RNAlevel or (FIG. 1F) by Western blotting at the protein level from CD8⁺ Tcell lysates. (FIG. 1G) Western blot showing upregulation of CTLA-4 byCD8⁺ T cells stimulated by IL-6. (FIG. 1H) IL-6 potently stimulatesCTLA-4 accumulation in the lipid rafts of CD8 T cells. Mouse primaryCD8⁺ T cells were stimulated for with IL-6. Single cell suspensions werestained for lipid rafts and CTLA-4 and analyzed by confocal microscopy(left panel). Lipid raft domains and CTLA-4 accumulations in lipid raftsupon IL-6 treatment (white arrowheads) are shown. Scale bar 2 μm.

FIGS. 2A-2F: CTLA-4^(apt)-Stat3siRNA improves the CD8⁺ T cell effectorresponse in vivo. (FIG. 2A) In vivo uptake of locally administeredCTLA-4^(apt.)Stat3siRNA by CD3⁺ (upper panel) and CD8⁺ (lower panel) Tcells from lymph nodes (LN) or tumor draining lymph nodes (TDLN),respectively. Single cell suspensions were analyzed by flow cytometry.(FIG. 2B) CTLA4^(apt)-siRNA^(FITC) positive and negative CD3⁺ T cells(CD3⁺FITC⁺ and CD3⁺FITC⁻) were isolated by FACS sorting from B16melanoma tumor-bearing mice. Expression of Stat3 mRNA was assessed byRT-PCR. SD shown. Two-tailed student's t-test performed and P-valueindicated. (FIG. 2C) In vivo knock-down efficiency ofCTLA-4^(apt.)Stat3siRNA. Flow cytometric analysis showing pStat3 levelsin CD8⁺ T cells from tumor draining lymph nodes postCTLA-4^(apt.)Stat3siRNA, CTLA-4^(apt.)LucsiRNA, or vehicle controltreatments. (FIG. 2D) Improved antigen-specific CD8⁺ T cell effectorfunction by CTLA-4^(apt.)Stat3siRNA CD8-OTI cells were adoptivelytransferred into B16-OVA tumor bearing Rag1^(−/−) mice. Mice weretreated four times every other day with CTLA-4^(apt.)Stat3siRNA,CTLA-4^(apt.)LucsiRNA, or left untreated after adoptive therapy. CD8⁺effector function was assessed by analyzing granzyme B (GrB) andinterferon γ (IFNγ) in ELISPOT assay after response recall withOVA^(SIINFEKL) peptide. SD shown; two-tailed student's t-test *) P<0.05,**) P<0.01, ***) P<0.001. (FIG. 2E) Analysis of improved T cell functionof lymphocytes isolated from tumor-draining lymph nodes after treatingB16 melanoma tumor bearing mice with CTLA-4^(apt.)Stat3siRNA orCTLA-4^(apt.)LucsiRNA. ELISPOT was performed after recalling the T cellresponse with B16 antigen-specific peptides p15E and TRP-2,respectively. SD shown; two-tailed student's t-test *) P<0.05, **)P<0.01, ***) P<0.001. (FIG. 2F) CD8⁺ T cell exhaustion was assessed byanalyzing PD-1 expression upon treating B16 tumor bearing mice withCTLA-4^(apt.)Stat3siRNA, CTLA-4^(apt.)LucsiRNA, or vehicle control (PBS)in flow cytometry.

FIGS. 3A-3G: CTLA-4^(apt.)Stat3siRNA is effective in inhibiting tumorgrowth and reducing tumor T_(Regs). (FIG. 3A) CTLA4^(apt.)Stat3siRNA vialocal administration reduces tumor T_(Reg). FoxP3-GFP⁺ T_(Reg) cellpopulation was imagined by in vivo multiphoton microscopy in B16melanoma tumors upon local treatments with the indicated siRNAconjugates, or vehicle control (PBS). ECM is given by 2HG. Scale bar 200μm. (FIG. 3B) Flow cytometry showing CD4⁺CD25⁺FoxP⁺ T_(Reg) cellpopulations in B16 melanoma tumors after treating with the indicatedsiRNA conjugates. (FIG. 3C) IL-10 production by GFP⁺ T_(Reg) cellsisolated from tumors of FoxP3-GFP transgenic mice treated as indicatedwas analyzed by flow cytometry. (FIG. 3D) Lung nodule formation wasdetermined in a lung colonization assay upon systemic administration ofB16 melanoma cells. Mice were treated systemically every other day asindicated. SD shown; two-tailed student's t-test *) P<0.05, **) P<0.01,***) P<0.001. (FIG. 3E) Homing of CD4⁺FoxP3⁺ T_(Reg) cells as well as(FIG. 3F) granzyme B⁺ CD8⁺ CTL infiltrating the lung was assessed inmicrosections of lungs after systemic administration of B16 melanomacells and systemic treatment of mice as indicated. Scale bar 100 μm. SDshown for CD8⁺ lung infiltrating cells; two-tailed student's t-test *)P<0.05, **) P<0.01, ***) P<0.001. (FIG. 3G) Growth kinetics for melanomatumor, renal cell carcinoma (Renca), lymphoma, and colon carcinoma wasassessed upon local administration of CTLA4^(apt)-conjugates or vehiclecontrol. Mice were treated every other day. SD shown; two-tailedstudent's t-test *) P<0.05, **) P<0.01, ***) P<0.001.

FIGS. 4A-4E: In vivo delivery of CTLA4-aptamer conjugate into CTLA4⁺human T cell lymphoma. (FIG. 4A) CTLA4 expression by Karpas299 human Tcell lymphoma was assessed by flow cytometry. (FIG. 4B) CTLA4-aptamer at500 pmol/ml and CTLA4 protein were analyzed for colocalization inKarpas299 T cell lymphoma cells by confocal microscopy in indicated timekinetics. Scale bar 10 μm. (FIG. 4C) CTLA4 protein recognition byCTLA4-aptamer. CTLA4-aptamer^(FITC) and CTLA4 protein were analyzed forinteraction upon aptamer treatment for 2 h in human T cell lymphoma byco-immunoprecipitation in a dose-dependent fashion as indicated. (FIG.4D) Flow cytometry analysis showing uptake kinetics of fluorescentCTLA-4^(apt.)STAT3siRNA by human Karpas299 T cell lymphoma at indicateddoses and time points in vitro. (FIG. 4E) Efficacy of in vivo silencingtargeting luciferase. Luciferase⁺ Karpas299 cells engraftedsubcutaneously in immune compromised mice were treated three times everyother day with CTLA4^(apt)-LuciferasesiRNA or CTLA4-aptamer as acontrol. Bioluminescent non-invasive imaging was performed time pointsas indicated and luminescent signal was quantified (right panel). SDshown; two-tailed student's t-test *) P<0.05, **) P<0.01, ***) P<0.001.

FIGS. 5A-5E: Treating human T cell lymphoma with CTLA4^(apt.)STAT3siRNAinhibits tumor growth. (FIG. 5A) Human T cell lymphoma tissue array wasstained for CTLA4 expression and analyzed by direct immunofluorescenceand (FIG. 5B) quantified. Magnified areas (dashed line) are shown in thelower left corner. Scale bar 100 μm. SD shown; two-tailed student'st-test *) P<0.05, **) P<0.01, ***) P<0.001. (FIG. 5C) Tumor growthkinetics of Karpas299 T cell lymphoma engrafted into athymic nude micetreated every other day with CTLA-4^(apt.)STAT3siRNA,CTLA-4^(apt.)LucsiRNA or vehicle (PBS). (FIG. 5D) Flow cytometry showingphospho-STAT3 expression and apoptotic cell death in the human T celllymphoma tumors treated as indicated. (FIG. 5E) Human T cell lymphomatumor microsections prepared from mice treated as indicated were stainedfor Ki67⁺ proliferative activity (left upper panels), CD31⁺ tumorvasculature (left middle panels), and B7-H1⁺ (left lower panels). Scalebar 100 μm. Fluorescent signals of Ki67 and B7-H1, and CD31⁺ bloodvessel length assessed by confocal microscopy were quantified (rightpanels) from independent FOV; bar graph shown CTLA-4^(apt.)STAT3siRNA(light gray), CTLA-4^(apt.)LucsiRNA (dark grey) or vehicle (PBS, black).SD shown; two-tailed student's t-test *) P<0.05, **) P<0.01, ***)P<0.001.

FIGS. 6A-6C: CTLA4^(apt) design and uptake in vitro and in vivo. (FIG.6A) Scheme of the CTLA4^(apt) with Stat3siRNA conjugate. (FIG. 6B) Forin vitro uptake, total nucleated splenocytes from wild-type C57BL/6 micewere pre-stained with fluorophore-conjugated surface markers (CD4, CD8,CD19, and CD11b) and then 1×10⁶ cells were treated with control(untreated) or FITC-conjugated CTLA4^(apt)-Stat3siRNA (500 pmol/ml) for2 hrs and then analyzed immediately by flow cytometry. (FIG. 6C) For invivo uptake, mice bearing established CT26 tumors were treated for 3consecutive days with control (untreated), unconjugatedCTLA4^(apt)-LucsiRNA, or FITC-conjugated CTLA4^(apt)-Stat3siRNA.Single-cell suspensions from tumors were analyzed for FITC-expressingcells by flow cytometry using surface markers for CD45, CD3, F4/80,CD11c, and CD19. Sequence legend: gggagagagg aagagggaug ggccgacgugccgcacgcgc uagaguacc (SEQ ID NO:4); gguacucuag cgcg (SEQ ID NO:5);caggguguca gaucacaugg gcuaa (SEQ ID NO:6); and uuagcccaug ugaucugacacccugaa (SEQ ID NO:7).

FIGS. 7A-7F: Induced T cell tolerance in IL-6 overexpressing tumors.(FIG. 7A) Murine fibrosarcoma 8101Re and 8101Pro were analyzed forcytokine expression profiles by cytokine arrays. (FIG. 7B) Elevated IL-6expression by fibrosarcoma 8101Pro was confirmed by RT-PCR (left) andELISA (right). (FIG. 7C) Fibrosarcoma 8101Re and 8101Pro were engraftedin syngeneic C57BL/6 mice and tumor growth kinetics were monitored. SDshown; two-tailed student's t-test *) P<0.05, **) P<0.01, ***) P<0.001.(FIG. 7D) Induction of CD8 T cell tolerance was assessed by monitoringtumor growth of fibrosarcoma 8101Re and 8101Pro engrafted in Rag1^(−/−)mice which received naïve CD8 T cells adoptively transferred when tumorsreached a volume of 300 mm³ (arrowhead). While 8101Re showed regressionupon T cell transfer, 8101Pro growth kinetics relapsed after a shortdelay (day 15-23). SD shown; two-tailed student's t-test *) P<0.05, **)P<0.01, ***) P<0.001. (FIG. 7E) Tumor infiltration of adoptivelytransferred CD8 T cells was visualized by confocal microscopy (left) andquantified (right). SD shown; two-tailed student's t-test *) P<0.05, **)P<0.01, ***) P<0.001. (FIG. 7F) Tumor-induced T cell tolerance wasassessed by adoptive T cell transfer to B16 melanoma bearing micecomparing antitumor activity of CD8 cells isolated from tumor bearingdonor mice (triangles) to CD8 cells isolated from naive donor mice(squares). Untreated mice were monitored as control (circles). SD shown;two-tailed student's t-test *) P<0.05, **) P<0.01, ***) P<0.001.

FIGS. 8A-8B: CTLA4 gene and its conservation. (FIG. 8A) Scheme of CTLA4gene full length showing exons 1-2, which represent the extracellulardomain (ECD), the transmembrane spanning region coded by exon 3, and thecytoplasmic tail coded by exon 4. (FIG. 8B) The amino acid sequence ofthe ligand binding region (B7 binding motif) of mouse and human CTLA4 ishighly conserved. (adapted from Teft, W. A. et al., Annu Rev Immunol24:65-97 (2006)). Sequence legend: QGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVID(SEQ ID NO:2); QGLRAVDTGLYLCKVELMYPPPYYVGMGNGTQIYVID (SEQ ID NO:3).

FIG. 9: Blood glucose reduction by treatment with CTLA-4apt-STAT3siRNA.Mice maintained on high-fat diet were treated systemically (via i.v.injection) with CTLA-4apt-STAT3siRNA, CTLA-4apt-LuciferasesiRNA for atleast 1 week or left untreated. Improved glucose tolerance was assessedfrom blood samples for time-points as indicated. Mice were fasted for 16h, 1 g per kg body weight of glucose injected i.p., and glucose levelswere measured every 15-30 min.

FIGS. 10A-10G: B cell lymphoma treatment with CTLA-4apt-STAT3siRNA.(FIG. 10A) B cell lymphoma express elevated levels of CTLA4 proteinassessed from patient biopsies. (FIG. 10B) CTLA4 protein locates to thecell surface in various human B cell lymphoma as shown by colocalizationstudies with surface marker CD19 in various human patient biopsies.Surface area is shown magnified (right panels). (FIG. 10C) Human B celllymphoma cell line Ly3, used as a model to study CTLA-4apt-STAT3siRNAtreatment efficacy, overexpress CTLA4 protein as shown by flowcytometry. (FIG. 10D) Efficient uptake of CTLA-4apt-STAT3siRNA dependenton CTLA4 expression was analyzed by flow cytometry. (FIG. 10E) Dose- andtime-dependent cellular internalization of CTLA-4apt-STAT3siRNA by Ly3cells was determined by flow cytometry. Cellular internalization ofCTLA-4apt-STAT3siRNA was acquired by confocal microscopy. Scale bar 10μm. (FIG. 10F) STAT3 silencing efficacy mediated by CTLA-4apt-STAT3siRNAwas analyzed in a dose-dependent fashion by flow cytometry as well as(FIG. 10G) demonstrated by STAT3 DNA binding activity shown by EMSA.

FIGS. 11A-11I: CTLA4 is overexpressed in human patients. FIG. 11A showsa staining against CTLA4 in a normal lymphnode and three types of B celllymphoma (Hodgkin's, small B cell lymphoma and diffuse B cell lymphoma).CTLA4 is not present in normal lymphnodes. In Hodgkin's, small B celllymphoma and diffuse B cell lymphoma CTLA4 is overexpressed. Therefore aCTLA4 overexpression is a common event in B cell lymphoma and CTLA4 is atarget in B cell lymphoma cells. The scale bar represents 100 μm. Lowerpanel: The lower panel shows a numeric quantification representing thedata of the upper panel in CTLA4+ area in percent. **: p=0.005. FIG.11B: CTLA4 is presented at the cell surface in human patients. FIG. 11Bshows a fluorescence microscopy analysis of Hodgkin's, ABC and Burkitt Bcell lymphomas. CTLA4 and CD19 (surface marker for B cells) staining.The small white dotted squares in the left panels are magnified on theright side. Therefore CTLA4 is functionally localized on the surface ofB cell lymphoma cells. The scale bar represents 10 μm. FIG. 11C: CTLA4expression in B cell lymphoma cell line Ly3. FIG. 11C shows a flowcytometry analysis of Ly3 B-cell lymphoma cells. The Ly3 cells werestained against CTLA4. As controls were used blank (solid grey)unstained cells and a 2nd antibody (black) staining. The X-axisrepresents the percentage of signal intensity (% of Max.) and the Y-axisrepresents the wavelength scale for the CTLA4 staining. FIG. 11D:CTLA4apt-STAT3siRNAFITC accumulates in B cell lymphoma cells with highCTLA4 expression. FIG. 11D shows a flow cytometry analysis of the B celllymphoma cell line Ly3 with and without a treatment of theCTLA4apt-STAT3siRNAFITC. This cell line has two populations of CTLA4expression (high and low). The CTLA4high population with no treatmentwas used as control (grey solid area). Therefore CTLA4apt-STAT3siRNAFITConly binds to cells with a high CTLA4 expression. This finding can beused as a tool for manipulation in B cell lymphoma cells with high CTLA4expression. FIG. 11E: CTLA4apt-STAT3siRNAFITC uptake after 120 min. and1,000 pmol. FIG. 11E shows a flow cytometry analysis of a kinetic uptakeexperiment. It has been shown, that 55% of CTLA4 positive B celllymphoma cells are loaded after 120 minutes of incubation with 500pmo1CTLA4apt-STAT3siRNAFITC. With 1,000 pmol CTLA4apt-STAT3siRNAFITC CTLA4positive B cell lymphoma cells are loaded up to 70% withCTLA4apt-STAT3siRNAFITC . Therefore we can specifically load andmanipulate CTLA4 positive B cell lymphoma cells withCTLA4apt-STAT3siRNAFITC. Right panel: CTLA4apt-STAT3siRNAFITCinternalizes into B cell lymphoma cells. The right panel shows amicroscopic (DIC) picture of the cells used in the left panel. FIG. 11F:STAT3 expression decreases by CTLA4apt-STAT3siRNAFITC treatment. FIG.11F shows in a flow cytometry analysis the pSTAT3 expression after aCTLA4apt-STAT3siRNAFITC treatment in Ly3 cells. After a 72 h treatmentwith 500 pmol CTLA4apt-STAT3siRNAFITC the pSTAT3 expression is reduced.FIG. 11G: STAT3 DNA binding decreases by CTLA4apt-STAT3siRNAFITCtreatment. FIG. 11F shows an EMSA experiment and point out the decreasedDNA binding of STAT3 after a CTLA4apt-STAT3siRNAFITC treatment. FIG.11H: CTLA4apt-STAT3siRNAFITC uptake after 15 min. in multiple myelomacells. FIG. 11H shows a flow cytometry analysis of an uptake experimentin the multiple myeloma cell line H929. It has been shown, that multiplemyeloma cells show a significant uptake of CTLA4apt-STAT3siRNAFITC after15 min. of treatment with 500 pmol. Therefore multiple myeloma cells arealso a target for CTLA4apt-STAT3siRNAFITC. FIG. 11I:CTLA4apt-STAT3siRNAFITC is processed by Dicer after 2 h in multiplemyeloma cells. FIG. 11I shows a fluorescence microscopy analysis of themultiple myeloma cell line H929 after a CTLA4apt-STAT3siRNAFITC. Theleft upper panel shows cells with no treatment, the right upper panelcells after 2h of treatment. The small white dotted square in the rightupper panels is magnified in the lower panel. The circles in both lowerpictures are located at the same position and show in the lower leftpanel CTLA4apt-STAT3siRNAFITC, in the lower right panel Dicer. ThereforeCTLA4apt-STAT3siRNAFITC and Dicer are co localized. The scale barrepresents 100 μm

FIGS. 12A-12C: Tumor growth with CTLA4apt-STAT3siRNAFITC treatment. FIG.12A: Local treatments of human B cell lymphoma tumors withCTLA4apt-STAT3siRNAFITC show a significant decrease tumor growth invivo. FIG. 12A shows the tumor growth curve of the in vivo experimentwith Ly3 (2×106 cells) engrafted human B cell lymphoma tumors. After 13days of growth the tumors were locally treated every other day withCTLA4apt-STAT3siRNAFITC (curve with triangles) andCTLA4apt-Luciferase-siRNA (curve with squares) (782.5 pmol/dose). Thevehicle is represented by the curve with diamonds. The x axis representsdays they axis the tumor volume. *: p=0.05, **: p=0.005. FIG. 12B:Systemic treatments of human B cell lymphoma tumors withCTLA4apt-STAT3siRNAFITC show a significant decreased tumor growth invivo. FIG. 12B shows the tumor growth curve of the in vivo experimentwith Ly3 (2×106 cells) engrafted human B cell lymphoma tumors. After 2days of growth the tumors were systemically treated every other day with782.5 pmol/dose CTLA4apt-STAT3siRNAFITC (curve with triangles) and391.25 pmol/dose CTLA4apt-STAT3siRNAFITC(curve with squares). Thevehicle is represented by the curve with diamonds. The x axis representsdays the y axis the tumor volume in mm3. *: p=0.05, **: p=0.005. FIG.12C: Local treatments of human B cell lymphoma tumors withCTLA4apt-STAT3siRNAFITC show significant effects in pSTAT3 expression,blood vessel length, amount of cl. caspase 3, Bc1XL expression and cellgrowth in vivo. FIG. 12C shows a fluorescence microscopy analysis oflocally treated tumors from the experiment in FIG. 2A. The OCT slideswere fixed with 2% paraformaldehyde (15 min.) followed by ice-coldmethanol (10 min.). After washing with PBS and an image enhancerincubation (30 min.) the slides were blocked for 1 h at room temperaturewith 10% goat, 2.5% mouse and 2.5% donkey serum in PBS. Following anovernight incubation with the primary antibodies (pSTAT3, CD31,cl.caspase 3, BclXL and Ki67 all 1:50) at 4° C. in a wet chamber theslides were washed and incubated with the secondary antibody for 30 min.pSTAT3, CD31 and cl. caspase 3 is represented in red, BclXL and Ki67 isrepresented in green and nuclei are represented in blue. After a finalwashing step the slides were mounted with Mowiol® 4-88 mounting media,stored for one night at 4° C. and analyzed via fluorescence microscopy.The scale bar represents 100 μm. Right panels: The right panels shows anumeric quantification representing the data of the left panels in MFIand μm. ***: p=0.0005

DETAILED DESCRIPTION OF THE INVENTION Definitions

While various embodiments and aspects of the present invention are shownand described herein, it will be obvious to those skilled in the artthat such embodiments and aspects are provided by way of example only.Numerous variations, changes, and substitutions will now occur to thoseskilled in the art without departing from the invention. It should beunderstood that various alternatives to the embodiments of the inventiondescribed herein may be employed in practicing the invention.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in the applicationincluding, without limitation, patents, patent applications, articles,books, manuals, and treatises are hereby expressly incorporated byreference in their entirety for any purpose.

The abbreviations used herein have their conventional meaning within thechemical and biological arts. The chemical structures and formulae setforth herein are constructed according to the standard rules of chemicalvalency known in the chemical arts.

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by a person of ordinaryskill in the art. See, e.g., Singleton et al., DICTIONARY OFMICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York,N.Y. 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL,Cold Springs Harbor Press (Cold Springs Harbor, N.Y. 1989). Any methods,devices and materials similar or equivalent to those described hereincan be used in the practice of this invention. The following definitionsare provided to facilitate understanding of certain terms usedfrequently herein and are not meant to limit the scope of the presentdisclosure.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e., unbranched) or branchednon-cyclic carbon chain (or carbon), or combination thereof, which maybe fully saturated, mono- or polyunsaturated and can include di- andmultivalent radicals, having the number of carbon atoms designated(i.e., C₁-C₁₀ means one to ten carbons). Examples of saturatedhydrocarbon radicals include, but are not limited to, groups such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, (cyclohexyl)methyl, homologs and isomers of, for example,n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkylgroup is one having one or more double bonds or triple bonds. Examplesof unsaturated alkyl groups include, but are not limited to, vinyl,2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl,3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and thehigher homologs and isomers. An alkoxy is an alkyl attached to theremainder of the molecule via an oxygen linker (—O—).

The term “alkylene,” by itself or as part of another substituent, means,unless otherwise stated, a divalent radical derived from an alkyl, asexemplified, but not limited by, —CH₂CH₂CH₂CH₂—. Typically, an alkyl (oralkylene) group will have from 1 to 24 carbon atoms, with those groupshaving 10 or fewer carbon atoms being preferred in the presentinvention. A “lower alkyl” or “lower alkylene” is a shorter chain alkylor alkylene group, generally having eight or fewer carbon atoms. Theterm “alkenylene,” by itself or as part of another substituent, means,unless otherwise stated, a divalent radical derived from an alkene.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable non-cyclic straight or branchedchain, or combinations thereof, including at least one carbon atom andat least one heteroatom selected from the group consisting of O, N, P,Si, and S, and wherein the nitrogen and sulfur atoms may optionally beoxidized, and the nitrogen heteroatom may optionally be quaternized. Theheteroatom(s) O, N, P, S, and Si may be placed at any interior positionof the heteroalkyl group or at the position at which the alkyl group isattached to the remainder of the molecule. Examples include, but are notlimited to: —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃,—CH₂—S—CH₂—CH₃, —CH₂—CH₂, —S(O)—CH₃,—CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃,—Si(CH₃)₃, —CH₂—CH═N—OCH₃, —CH═CH—N(CH₃)—CH₃, —O—CH₃, —O—CH₂—CH₃, and—CN. Up to two or three heteroatoms may be consecutive, such as, forexample, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃.

Similarly, the term “heteroalkylene,” by itself or as part of anothersubstituent, means, unless otherwise stated, a divalent radical derivedfrom heteroalkyl, as exemplified, but not limited by,—CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylenegroups, heteroatoms can also occupy either or both of the chain termini(e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, andthe like). Still further, for alkylene and heteroalkylene linkinggroups, no orientation of the linking group is implied by the directionin which the formula of the linking group is written. For example, theformula —C(O)₂R′— represents both —C(O)₂R′— and —R′C(O)₂—. As describedabove, heteroalkyl groups, as used herein, include those groups that areattached to the remainder of the molecule through a heteroatom, such as—C(O)R′, —C(O)NR′, —NR′R″, —OR′, —SR′, and/or —SO₂R′. Where“heteroalkyl” is recited, followed by recitations of specificheteroalkyl groups, such as —NR′R″ or the like, it will be understoodthat the terms heteroalkyl and —NR′R″ are not redundant or mutuallyexclusive. Rather, the specific heteroalkyl groups are recited to addclarity. Thus, the term “heteroalkyl” should not be interpreted hereinas excluding specific heteroalkyl groups, such as —NR′R″ or the like.

The terms “cycloalkyl” and “heterocycloalkyl,” by themselves or incombination with other terms, mean, unless otherwise stated, cyclicnon-aromatic versions of “alkyl” and “heteroalkyl,” respectively,wherein the carbons making up the ring or rings do not necessarily needto be bonded to a hydrogen due to all carbon valencies participating inbonds with non-hydrogen atoms. Additionally, for heterocycloalkyl, aheteroatom can occupy the position at which the heterocycle is attachedto the remainder of the molecule. Examples of cycloalkyl include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. A “cycloalkylene” and a“heterocycloalkylene,” alone or as part of another substituent, means adivalent radical derived from a cycloalkyl and heterocycloalkyl,respectively.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” includes, but is not limited to, fluoromethyl,difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl,3-bromopropyl, and the like.

The term “acyl” means, unless otherwise stated, —C(O)R where R is asubstituted or unsubstituted alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent, which can be a single ring ormultiple rings (preferably from 1 to 3 rings) that are fused together(i.e., a fused ring aryl) or linked covalently (e.g., biphenyl). A fusedring aryl refers to multiple rings fused together wherein at least oneof the fused rings is an aryl ring. The term “heteroaryl” refers to arylgroups (or rings) that contain at least one heteroatom such as N, O, orS, wherein the nitrogen and sulfur atoms are optionally oxidized, andthe nitrogen atom(s) are optionally quaternized. Thus, the term“heteroaryl” includes fused ring heteroaryl groups (i.e., multiple ringsfused together wherein at least one of the fused rings is aheteroaromatic ring). A 5,6-fused ring heteroarylene refers to two ringsfused together, wherein one ring has 5 members and the other ring has 6members, and wherein at least one ring is a heteroaryl ring. Likewise, a6,6-fused ring heteroarylene refers to two rings fused together, whereinone ring has 6 members and the other ring has 6 members, and wherein atleast one ring is a heteroaryl ring. And a 6,5-fused ring heteroarylenerefers to two rings fused together, wherein one ring has 6 members andthe other ring has 5 members, and wherein at least one ring is aheteroaryl ring. A heteroaryl group can be attached to the remainder ofthe molecule through a carbon or heteroatom. Non-limiting examples ofaryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl,4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below. An “arylene” and a“heteroarylene,” alone or as part of another substituent, mean adivalent radical derived from an aryl and heteroaryl, respectively.Non-limiting examples of heteroaryl groups include pyridinyl,pyrimidinyl, thiophenyl, thienyl, furanyl, indolyl, benzoxadiazolyl,benzodioxolyl, benzodioxanyl, thianaphthanyl, pyrrolopyridinyl,indazolyl, quinolinyl, quinoxalinyl, pyridopyrazinyl, quinazolinonyl,benzoisoxazolyl, imidazopyridinyl, benzofuranyl, benzothienyl,benzothiophenyl, phenyl, naphthyl, biphenyl, pyrrolyl, pyrazolyl,imidazolyl, pyrazinyl, oxazolyl, isoxazolyl, thiazolyl, furylthienyl,pyridyl, pyrimidyl, benzothiazolyl, purinyl, benzimidazolyl,isoquinolyl, thiadiazolyl, oxadiazolyl, pyrrolyl, diazolyl, triazolyl,tetrazolyl, benzothiadiazolyl, isothiazolyl, pyrazolopyrimidinyl,pyrrolopyrimidinyl, benzotriazolyl, benzoxazolyl, or quinolyl. Theexamples above may be substituted or unsubstituted and divalent radicalsof each heteroaryl example above are non-limiting examples ofheteroarylene.

A fused ring heterocyloalkyl-aryl is an aryl fused to aheterocycloalkyl. A fused ring heterocycloalkyl-heteroaryl is aheteroaryl fused to a heterocycloalkyl. A fused ringheterocycloalkyl-cycloalkyl is a heterocycloalkyl fused to a cycloalkyl.A fused ring heterocycloalkyl-heterocycloalkyl is a heterocycloalkylfused to another heterocycloalkyl. Fused ring heterocycloalkyl-aryl,fused ring heterocycloalkyl-heteroaryl, fused ringheterocycloalkyl-cycloalkyl, or fused ringheterocycloalkyl-heterocycloalkyl may each independently beunsubstituted or substituted with one or more of the substitutentsdescribed herein.

The term “oxo,” as used herein, means an oxygen that is double bonded toa carbon atom.

The term “alkylsulfonyl,” as used herein, means a moiety having theformula —S(O₂)—R′, where R′ is a substituted or unsubstituted alkylgroup as defined above. R′ may have a specified number of carbons (e.g.,“C₁-C₄ alkylsulfonyl”).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl,” and“heteroaryl”) includes both substituted and unsubstituted forms of theindicated radical. Preferred substituents for each type of radical areprovided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,—halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)N R′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —NR′NR″R′″,—ONR′R″, —NR′C═(O)NR″NR′″R″″, —CN, —NO₂, in a number ranging from zeroto (2m′+1), where m′ is the total number of carbon atoms in suchradical. R, R′, R″, R′″, and R″″ each preferably independently refer tohydrogen, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl (e.g., aryl substituted with 1-3halogens), substituted or unsubstituted heteroaryl, substituted orunsubstituted alkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups.When a compound of the invention includes more than one R group, forexample, each of the R groups is independently selected as are each R′,R″, R′″, and R″″ group when more than one of these groups is present.When R′ and R″ are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 4-, 5-, 6-, or 7-memberedring. For example, —NR′R″ includes, but is not limited to,1-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound togroups other than hydrogen groups, such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and thelike).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, for example: —OR′, —NR′R″, —SR′, —halogen, —SiR′R″R′″,—OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC (O)NR′R″, —NR″C(O)R′,—NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″,—S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —NR′NR″R′″, —ONR′R″,—NR′C═(O)NR″NR′″R″″, —CN, —NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl, in a number ranging from zero tothe total number of open valences on the aromatic ring system; and whereR′, R″, R′″, and R″″ are preferably independently selected fromhydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl. When acompound of the invention includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″,and R″″ groups when more than one of these groups is present.

Two or more substituents may optionally be joined to form aryl,heteroaryl, cycloalkyl, or heterocycloalkyl groups. Such so-calledring-forming substituents are typically, though not necessarily, foundattached to a cyclic base structure. In one embodiment, the ring-formingsubstituents are attached to adjacent members of the base structure. Forexample, two ring-forming substituents attached to adjacent members of acyclic base structure create a fused ring structure. In anotherembodiment, the ring-forming substituents are attached to a singlemember of the base structure. For example, two ring-forming substituentsattached to a single member of a cyclic base structure create aspirocyclic structure. In yet another embodiment, the ring-formingsubstituents are attached to non-adjacent members of the base structure.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally form a ring of the formula —T—C(O)—(CRR′)_(q)—U—, whereinT and U are independently —NR—, —O—, —CRR′—, or a single bond, and q isan integer of from 0 to 3. Alternatively, two of the substituents onadjacent atoms of the aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -A-(CH₂)₁—B—, wherein A and B areindependently —CRR′—, —O—, —NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′—, or asingle bond, and r is an integer of from 1 to 4. One of the single bondsof the new ring so formed may optionally be replaced with a double bond.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula —(CRR′)_(s)—X′— (C″R″R′″)_(d)—, where s and d are independentlyintegers of from 0 to 3, and X′ is —O—, —S—, —S(O)—, —S(O)₂—, or—S(O)₂NR′—. The substituents R, R′, R″, and R′″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl.

As used herein, the terms “heteroatom” or “ring heteroatom” are meant toinclude, oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), andsilicon (Si).

A “substituent group,” as used herein, means a group selected from thefollowing moieties:

-   -   (A) oxo, halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,        —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,        —NHC═(O)NHNH₂, —NHC═(O) NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH,        —NHOH, —OCF₃, —OCHF₂, —NHSO₂CH₃, —N₃, unsubstituted alkyl,        unsubstituted heteroalkyl, unsubstituted cycloalkyl,        unsubstituted heterocycloalkyl, unsubstituted aryl,        unsubstituted heteroaryl, and    -   (B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,        heteroaryl, substituted with at least one substituent selected        from:        -   (i) oxo, halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,            —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,            —NHC═(O)NHNH₂, —NHC═(O) NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH,            —NHOH, —OCF₃, —OCHF₂, —NHSO₂CH₃, —N₃, unsubstituted alkyl,            unsubstituted heteroalkyl, unsubstituted cycloalkyl,            unsubstituted heterocycloalkyl, unsubstituted aryl,            unsubstituted heteroaryl, and        -   (ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,            heteroaryl, substituted with at least one substituent            selected from:            -   (a) oxo, halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂,                —NO₂, —SH, —SO₂Cl, —SO₀₃H, —SO₄H, —SO₂NH₂, —NHNH₂,                —ONH₂, —NHC═(O)NHNH₂, —NHC═(O) NH₂, —NHSO₂H, —NHC═(O)H,                —NHC(O)—OH, —NHOH, —OCF₃, —OCHF₂, —NHSO₂CH₃, —N₃,                unsubstituted alkyl, unsubstituted heteroalkyl,                unsubstituted cycloalkyl, unsubstituted                heterocycloalkyl, unsubstituted aryl, unsubstituted                heteroaryl, and            -   (b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,                aryl, heteroaryl, substituted with at least one                substituent selected from: oxo, halogen, —CF₃, —CN, —OH,                —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H,                —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂, —NHC═(O) NH₂,                —NHSO₂H, —NHC═(O)H, —NHC(O)—OH, —NHOH, —OCF₃, —OCHF₂,                —NHSO₂CH₃, —N₃, unsubstituted alkyl, unsubstituted                heteroalkyl, unsubstituted cycloalkyl, unsubstituted                heterocycloalkyl, unsubstituted aryl, unsubstituted                heteroaryl.

A “size-limited substituent” or “size-limited substituent group,” asused herein, means a group selected from all of the substituentsdescribed above for a “substituent group,” wherein each substituted orunsubstituted alkyl is a substituted or unsubstituted C₁-C₂₀ alkyl, eachsubstituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈cycloalkyl, each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 3 to 8 membered heterocycloalkyl, eachsubstituted or unsubstituted aryl is a substituted or unsubstitutedC₆-C₁₀ aryl, and each substituted or unsubstituted heteroaryl is asubstituted or unsubstituted 5 to 10 membered heteroaryl.

A “lower substituent” or “lower substituent group,” as used herein,means a group selected from all of the substituents described above fora “substituent group,” wherein each substituted or unsubstituted alkylis a substituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₃-C₇ cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7membered heterocycloalkyl, each substituted or unsubstituted aryl is asubstituted or unsubstituted C₆-C₁₀ aryl, and each substituted orunsubstituted heteroaryl is a substituted or unsubstituted 5 to 9membered heteroaryl.

In some embodiments, each substituted group described in the compoundsherein is substituted with at least one substituent group. Morespecifically, in some embodiments, each substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl, substituted heteroaryl, substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene described in the compounds herein are substituted with atleast one substituent group. In other embodiments, at least one or allof these groups are substituted with at least one size-limitedsubstituent group. In other embodiments, at least one or all of thesegroups are substituted with at least one lower substituent group.

In other embodiments of the compounds herein, each substituted orunsubstituted alkyl may be a substituted or unsubstituted C₁-C₂₀ alkyl,each substituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈cycloalkyl, each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 3 to 8 membered heterocycloalkyl, eachsubstituted or unsubstituted aryl is a substituted or unsubstitutedC₆-C₁₀ aryl, and/or each substituted or unsubstituted heteroaryl is asubstituted or unsubstituted 5 to 10 membered heteroaryl. In someembodiments of the compounds herein, each substituted or unsubstitutedalkylene is a substituted or unsubstituted C₁-C₂₀ alkylene, eachsubstituted or unsubstituted heteroalkylene is a substituted orunsubstituted 2 to 20 membered heteroalkylene, each substituted orunsubstituted cycloalkylene is a substituted or unsubstituted C₃-C₈cycloalkylene, each substituted or unsubstituted heterocycloalkylene isa substituted or unsubstituted 3 to 8 membered heterocycloalkylene, eachsubstituted or unsubstituted arylene is a substituted or unsubstitutedC₆-C₁₀ arylene, and/or each substituted or unsubstituted heteroaryleneis a substituted or unsubstituted 5 to 10 membered heteroarylene.

In some embodiments, each substituted or unsubstituted alkyl is asubstituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₃-C₇ cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7membered heterocycloalkyl, each substituted or unsubstituted aryl is asubstituted or unsubstituted C₆-C₁₀ aryl, and/or each substituted orunsubstituted heteroaryl is a substituted or unsubstituted 5 to 9membered heteroaryl. In some embodiments, each substituted orunsubstituted alkylene is a substituted or unsubstituted C₁-C₈ alkylene,each substituted or unsubstituted heteroalkylene is a substituted orunsubstituted 2 to 8 membered heteroalkylene, each substituted orunsubstituted cycloalkylene is a substituted or unsubstituted C₃-C₇cycloalkylene, each substituted or unsubstituted heterocycloalkylene isa substituted or unsubstituted 3 to 7 membered heterocycloalkylene, eachsubstituted or unsubstituted arylene is a substituted or unsubstitutedC₆-C₁₀ arylene, and/or each substituted or unsubstituted heteroaryleneis a substituted or unsubstituted 5 to 9 membered heteroarylene. In someembodiments, the compound is a chemical species set forth in theExamples section below.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form, orcomplements thereof. The term “polynucleotide” refers to a linearsequence of nucleotides. The term “nucleotide” typically refers to asingle unit of a polynucleotide, i.e., a monomer. Nucleotides can beribonucleotides, deoxyribonucleotides, or modified versions thereof.Examples of polynucleotides contemplated herein include single anddouble stranded DNA, single and double stranded RNA (including siRNA),and hybrid molecules having mixtures of single and double stranded DNAand RNA. The terms also encompass nucleic acids containing knownnucleotide analogs or modified backbone residues or linkages, which aresynthetic, naturally occurring, and non-naturally occurring, which havesimilar binding properties as the reference nucleic acid, and which aremetabolized in a manner similar to the reference nucleotides. Examplesof such analogs include, without limitation, phosphodiester derivativesincluding, e.g., phosphoramidate, phosphorodiamidate, phosphorothioate(also known as phosphothioate), phosphorodithioate, phosphonocarboxylicacids, phosphonocarboxylates, phosphonoacetic acid, phosphonoformicacid, methyl phosphonate, boron phosphonate, or O-methylphosphoroamiditelinkages (see Eckstein, Oligonucleotides and Analogues: A PracticalApproach, Oxford University Press); and peptide nucleic acid backbonesand linkages. Other analog nucleic acids include those with positivebackbones; non-ionic backbones, modified sugars, and non-ribosebackbones (e.g. phosphorodiamidate morpholino oligos or locked nucleicacids (LNA)), including those described in U.S. Pat. Nos. 5,235,033 and5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CarbohydrateModifications in Antisense Research, Sanghui & Cook, eds. Nucleic acidscontaining one or more carbocyclic sugars are also included within onedefinition of nucleic acids. Modifications of the ribose-phosphatebackbone may be done for a variety of reasons, e.g., to increase thestability and half-life of such molecules in physiological environmentsor as probes on a biochip. Mixtures of naturally occurring nucleic acidsand analogs can be made; alternatively, mixtures of different nucleicacid analogs, and mixtures of naturally occurring nucleic acids andanalogs may be made. In embodiments, the internucleotide linkages in DNAare phosphodiester, phosphodiester derivatives, or a combination ofboth.

The words “complementary” or “complementarity” refer to the ability of anucleic acid in a polynucleotide to form a base pair with anothernucleic acid in a second polynucleotide. For example, the sequence A-G-Tis complementary to the sequence T-C-A. Complementarity may be partial,in which only some of the nucleic acids match according to base pairing,or complete, where all the nucleic acids match according to basepairing.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are near each other, and, inthe case of a secretory leader, contiguous and in reading phase.However, enhancers do not have to be contiguous. Linking is accomplishedby ligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The term “probe” or “primer”, as used herein, is defined to be one ormore nucleic acid fragments whose specific hybridization to a sample canbe detected. A probe or primer can be of any length depending on theparticular technique it will be used for. For example, PCR primers aregenerally between 10 and 40 nucleotides in length, while nucleic acidprobes for, e.g., a Southern blot, can be more than a hundrednucleotides in length. The probe may be unlabeled or labeled asdescribed below so that its binding to the target or sample can bedetected. The probe can be produced from a source of nucleic acids fromone or more particular (preselected) portions of a chromosome, e.g., oneor more clones, an isolated whole chromosome or chromosome fragment, ora collection of polymerase chain reaction (PCR) amplification products.The length and complexity of the nucleic acid fixed onto the targetelement is not critical to the invention. One of skill can adjust thesefactors to provide optimum hybridization and signal production for agiven hybridization procedure, and to provide the required resolutionamong different genes or genomic locations.

The probe may also be isolated nucleic acids immobilized on a solidsurface (e.g., nitrocellulose, glass, quartz, fused silica slides), asin an array. In some embodiments, the probe may be a member of an arrayof nucleic acids as described, for instance, in WO 96/17958. Techniquescapable of producing high density arrays can also be used for thispurpose (see, e.g., Fodor (1991) Science 767-773; Johnston (1998) Curr.Biol. 8: R171-R174; Schummer (1997) Biotechniques 23: 1087-1092; Kern(1997) Biotechniques 23: 120-124; U.S. Pat. No. 5,143,854).

A “labeled nucleic acid probe or oligonucleotide” is one that is bound,either covalently, through a linker or a chemical bond, ornoncovalently, through ionic, van der Waals, electrostatic, or hydrogenbonds to a label such that the presence of the probe may be detected bydetecting the presence of the label bound to the probe. Alternatively, amethod using high affinity interactions may achieve the same resultswhere one of a pair of binding partners binds to the other, e.g.,biotin, streptavidin.

The term “gene” means the segment of DNA involved in producing aprotein; it includes regions preceding and following the coding region(leader and trailer) as well as intervening sequences (introns) betweenindividual coding segments (exons). The leader, the trailer as well asthe introns include regulatory elements that are necessary during thetranscription and the translation of a gene. Further, a “protein geneproduct” is a protein expressed from a particular gene.

The word “expression” or “expressed” as used herein in reference to agene means the transcriptional and/or translational product of thatgene. The level of expression of a DNA molecule in a cell may bedetermined on the basis of either the amount of corresponding mRNA thatis present within the cell or the amount of protein encoded by that DNAproduced by the cell. The level of expression of non-coding nucleic acidmolecules (e.g., siRNA) may be detected by standard PCR or Northern blotmethods well known in the art. See, Sambrook et al., 1989 MolecularCloning: A Laboratory Manual, 18.1-18.88.

An “inhibitory nucleic acid” is a nucleic acid (e.g. DNA, RNA, polymerof nucleotide analogs) that is capable of inhibiting the function of atarget protein (e.g., STAT3) by binding to a target nucleic acid (e.g.an mRNA translatable into STAT3) and reducing transcription of thetarget nucleic acid (e.g. mRNA from DNA) or reducing the translation ofthe target nucleic acid (e.g.mRNA) or altering transcript splicing. Inembodiments, the inhibitory nucleic acid is a nucleic acid that iscapable of binding (e.g. hybridizing) to a target nucleic acid (e.g. anmRNA translatable into an STAT3) and reducing translation of the targetnucleic acid. The target nucleic acid is or includes one or more targetnucleic acid sequences to which the inhibitory nucleic acid binds (e.g.hybridizes). In embodiments, an inhibitory nucleic acid is or includes asequence (also referred to herein as an “antisense nucleic acidsequence”) that is capable of hybridizing to at least a portion of atarget nucleic acid at a target nucleic acid sequence. An example of aninhibitory nucleic acid is an antisense nucleic acid. Thus, inembodiments, the inhibitory nucleic acid is an antisense nucleic acid.

In embodiments, the inhibitory nucleic acid is a ribozyme. A “ribozyme”as provided herein refers to a ribonucleic acid capable of enzymaticallymodifying RNA (e.g., cleaving, splicing).

In embodiments, the inhibitory nucleic acid is an RNA decoy. An “RNAdecoy” as provided herein is an RNA molecule, which inhibits thefunction of a protein (e.g., viral protein or cellular protein) bybinding the protein. The RNA decoy may inhibit protein function bypreventing the interaction between a target protein (e.g., HIV Tat) andits natural interaction partners (e.g., TAR). Further, the binding of anRNA decoy to a target protein may alter the subcellular location of thetarget protein thereby inhibiting its activity.

An “antisense nucleic acid” as referred to herein is a DNA or RNAmolecule that is complementary to at least a portion of a specifictarget nucleic acid (e.g. an mRNA translatable into a protein) and isacapable of reducing transcription of the target nucleic acid (e.g. mRNAfrom DNA) or reducing the translation of the target nucleic acid(e.g.mRNA) or altering transcript splicing (e.g. single strandedmorpholino oligo). See, e.g., Weintraub, Scientific American, 262:40(1990). Typically, synthetic antisense oligonucleotides are generallybetween 15 and 25 bases in length. Thus, antisense nucleic acids arecapable of hybridizing to (e.g.

selectively hybridizing to) a target nucleic acid (e.g. target mRNA). Inembodiments, the antisense nucleic acid hybridizes to the target nucleicacid sequence (e.g. mRNA) under stringent hybridization conditions. Inembodiments, the antisense nucleic acid hybridizes to the target nucleicacid (e.g. mRNA) under moderately stringent hybridization conditions.Antisense nucleic acids may comprise naturally occurring nucleotides ormodified nucleotides such as, e.g., phosphorothioate, methylphosphonate,and -anomeric sugar-phosphate, backbonemodified nucleotides.

In the cell, the antisense nucleic acids hybridize to the correspondingmRNA, forming a double-stranded molecule. The antisense nucleic acidsinterfere with the translation of the mRNA, since the cell will nottranslate an mRNA that is double-stranded. The use of antisense methodsto inhibit the in vitro translation of genes is well known in the art(Marcus-Sakura, Anal. Biochem., 172:289, (1988)). Further, antisensemolecules which bind directly to the DNA may be used. Antisense nucleicacids may be single or double stranded nucleic acids. Non-limitingexamples of antisense nucleic acids include siRNAs (including theirderivatives or pre-cursors, such as nucleotide analogs), short hairpinRNAs (shRNA), micro RNAs (miRNA) and small nucleolar RNAs (snoRNA) orcertain of their derivatives or pre-cursors.

A “siRNA,” “small interfering RNA,” “small RNA,” or “RNAi” as providedherein refers to a nucleic acid that forms a double stranded RNA, whichdouble stranded RNA has the ability to reduce or inhibit expression of agene or target gene (e.g. when expressed in the same cell as the gene ortarget gene). The complementary portions of the nucleic acid thathybridize to form the double stranded molecule typically havesubstantial or complete identity. In one embodiment, a siRNA or RNAi isa nucleic acid that has substantial or complete identity to a targetgene and forms a double stranded siRNA. In embodiments, the siRNAinhibits gene expression by interacting with a complementary cellularmRNA thereby interfering with the expression of the complementary mRNA.Typically, the nucleic acid is at least about 15-50 nucleotides inlength (e.g., each complementary sequence of the double stranded siRNAis 15-50 nucleotides in length, and the double stranded siRNA is about15-50 base pairs in length). In other embodiments, the length is 20-30base nucleotides, preferably about 20-25 or about 24-29 nucleotides inlength, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotidesin length.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all. Transgenic cells and plants are thosethat express a heterologous gene or coding sequence, typically as aresult of recombinant methods.

The term “exogenous” refers to a molecule or substance (e.g., acompound, nucleic acid or protein) that originates from outside a givencell or organism. For example, an “exogenous promoter” as referred toherein is a promoter that does not originate from the plant it isexpressed by. Conversely, the term “endogenous” or “endogenous promoter”refers to a molecule or substance that is native to, or originateswithin, a given cell or organism.

The term “isolated”, when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is essentially free of other cellularcomponents with which it is associated in the natural state. It can be,for example, in a homogeneous state and may be in either a dry oraqueous solution. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinthat is the predominant species present in a preparation issubstantially purified.

The term “aptamer” as provided herein refers to short oligonucleotides(e.g. deoxyribonucleotides), which fold into molecular structures thatbind with high affinity and specificity to proteins, peptides, and smallmolecules. Aptamers can be selected in vitro from very large librariesof randomized sequences by the process of systemic evolution of ligandsby exponential enrichment (SELEX as described in Ellington A D, SzostakJ W (1990) In vitro selection of RNA molecules that bind specificligands. Nature 346:818-822; Tuerk C, Gold L (1990) Systematic evolutionof ligands by exponential enrichment: RNA ligands to bacteriophage T4DNA polymerase. Science 249:505-510) or by developing SOMAmers (slowoff-rate modified aptamers) (Gold L et al. (2010) Aptamer-basedmultiplexed proteomic technology for biomarker discovery. PLoS ONE5(12):e15004). Applying the SELEX and the SOMAmer technology includesfor instance adding functional groups that mimic amino acid side chainsto expand the aptamer's chemical diversity. As a result high affinityaptamers for almost any protein target are enriched and identified.Aptamers exhibit many desirable properties for targeted drug delivery,such as ease of selection and synthesis, high binding affinity andspecificity, low immunogenicity, and versatile synthetic accessibility.To date, a variety of anti-cancer agents (e.g. chemotherapy drugs,toxins, and siRNAs) have been successfully delivered to cancer cells invitro using apatmers.

As used herein, the term “conjugated” when referring to two moietiesmeans the two moieties (e.g., a monomeric CLTA-4 aptamer nucleic acidand a cell activity modulating nucleic acid) are bonded, wherein thebond or bonds connecting the two moieties may be covalent ornon-covalent. In embodiments, the two moieties are covalently bonded toeach other (e.g. directly or through a covalently bonded intermediary).In embodiments, the two moieties are non-covalently bonded (e.g. throughionic bond(s), van der Waal's bond(s)/interactions, hydrogen bond(s),polar bond(s), or combinations or mixtures thereof).

As used herein, the term “about” means a range of values including thespecified value, which a person of ordinary skill in the art wouldconsider reasonably similar to the specified value. In embodiments, theterm “about” means within a standard deviation using measurementsgenerally acceptable in the art. In embodiments, about means a rangeextending to +/−10% of the specified value. In embodiments, about meansthe specified value.

The terms “protein”, “peptide”, and “polypeptide” are usedinterchangeably to denote an amino acid polymer or a set of two or moreinteracting or bound amino acid polymers. The terms apply to amino acidpolymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers and non-naturallyoccurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid. The terms“non-naturally occurring amino acid” and “unnatural amino acid” refer toamino acid analogs, synthetic amino acids, and amino acid mimetics whichare not found in nature.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence withrespect to the expression product, but not with respect to actual probesequences.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over aspecified region, when compared and aligned for maximum correspondenceover a comparison window or designated region) as measured using a BLASTor BLAST 2.0 sequence comparison algorithms with default parametersdescribed below, or by manual alignment and visual inspection (see,e.g., NCBI web site http://www.ncbi.nlm.nih.gov/BLAST/ or the like).Such sequences are then said to be “substantially identical.” Thisdefinition also refers to, or may be applied to, the compliment of atest sequence. The definition also includes sequences that havedeletions and/or additions, as well as those that have substitutions. Asdescribed below, the preferred algorithms can account for gaps and thelike. Preferably, identity exists over a region that is at least about25 amino acids or nucleotides in length, or more preferably over aregion that is 50-100 amino acids or nucleotides in length.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence with a higher affinity, e.g., under more stringentconditions, than to other nucleotide sequences (e.g., total cellular orlibrary DNA or RNA).

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acids, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tij ssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditions mayalso be achieved with the addition of destabilizing agents such asformamide. For selective or specific hybridization, a positive signal isat least two times background, preferably 10 times backgroundhybridization. Exemplary stringent hybridization conditions can be asfollowing: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or,5x SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDSat 65° C.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency. Additional guidelines for determininghybridization parameters are provided in numerous reference, e.g., andCurrent Protocols in Molecular Biology, ed. Ausubel, et al.

For specific proteins described herein (e.g., STAT3, FoxP3, CTLA-4), thenamed protein includes any of the protein's naturally occurring forms,or variants that maintain the protein transcription factor activity(e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%activity compared to the native protein). In some embodiments, variantshave at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequenceidentity across the whole sequence or a portion of the sequence (e.g. a50, 100, 150 or 200 continuous amino acid portion) compared to anaturally occurring form. In other embodiments, the protein is theprotein as identified by its NCBI sequence reference. In otherembodiments, the protein is the protein as identified by its NCBIsequence reference or functional fragment thereof

The term “CTLA-4” as provided herein includes any of the CytotoxicT-Lymphocyte Antigen 4 (CTLA-4) protein naturally occurring forms,homologs or variants that maintain the protein transcription factoractivity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%or 100% activity compared to the native protein). In some embodiments,variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100%amino acid sequence identity across the whole sequence or a portion ofthe sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion)compared to a naturally occurring form. In embodiments, the CTLA-4protein is the protein as identified by the NCBI sequence referenceGI:21361212. In embodiments, the CTLA-4 protein is the protein asidentified by the NCBI sequence reference GI:21361212 or functionalfragment thereof. In embodiments, the CTLA-4 protein includes thesequence of SEQ ID NO:2 or SEQ ID NO:3. In embodiments, the CTLA-4protein is encoded by a nucleic acid sequences corresponding to Gene ID:1493.

A “STAT3 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding Signal transducer andActivator of transcription 3 (STAT3), homologs or variants thereof thatmaintain STAT3 protein activity (e.g. within at least 50%, 80%, 90%,95%, 96%, 97%, 98%, 99% or 100% activity compared to STAT3). In someaspects, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100%amino acid sequence identity across the whole sequence or a portion ofthe sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion)compared to a naturally occurring STAT3 polypeptide. In embodiments, theSTAT3 gene is substantially identical to the nucleic acid identified bythe NCBI reference number Gene ID: 6774 or a variant having substantialidentity thereto.

A “FoxP3 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding Forkhead Box P3 (FoxP3),homologs or variants thereof that maintain FoxP3 protein activity (e.g.within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activitycompared to FoxP3). In some aspects, variants have at least 90%, 95%,96%, 97%, 98%, 99% or 100% amino acid sequence identity across the wholesequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring FoxP3polypeptide. In embodiments, the FoxP3 gene is substantially identicalto the nucleic acid identified by the NCBI reference number GeneID:50943 or a variant having substantial identity thereto.

“Anti-cancer agent” is used in accordance with its plain ordinarymeaning and refers to a composition (e.g. compound, drug, antagonist,inhibitor, modulator) having antineoplastic properties or the ability toinhibit the growth or proliferation of cells. In embodiments, ananti-cancer agent is a chemotherapeutic. In embodiments, an anti-canceragent is an agent identified herein having utility in methods oftreating cancer. In embodiments, an anti-cancer agent is an agentapproved by the FDA or similar regulatory agency of a country other thanthe USA, for treating cancer. Examples of anti-cancer agents include,but are not limited to, MEK (e.g. MEK1, MEK2, or MEK1 and MEK2)inhibitors (e.g. XL518, CI-1040, PD035901, selumetinib/AZD6244,GSK1120212/trametinib, GDC-0973, ARRY-162, ARRY-300, AZD8330, PD0325901,U0126, PD98059, TAK-733, PD318088, AS703026, BAY 869766), alkylatingagents (e.g., cyclophosphamide, ifosfamide, chlorambucil, busulfan,melphalan, mechlorethamine, uramustine, thiotepa, nitrosoureas, nitrogenmustards (e.g., mechloroethamine, cyclophosphamide, chlorambucil,meiphalan), ethylenimine and methylmelamines (e.g., hexamethlymelamine,thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g.,carmustine, lomusitne, semustine, streptozocin), triazenes(decarbazine)), anti-metabolites (e.g., 5-azathioprine, leucovorin,capecitabine, fludarabine, gemcitabine, pemetrexed, raltitrexed, folicacid analog (e.g., methotrexate), or pyrimidine analogs (e.g.,fluorouracil, floxouridine, Cytarabine), purine analogs (e.g.,mercaptopurine, thioguanine, pentostatin), etc.), plant alkaloids (e.g.,vincristine, vinblastine, vinorelbine, vindesine, podophyllotoxin,paclitaxel, docetaxel, etc.), topoisomerase inhibitors (e.g.,irinotecan, topotecan, amsacrine, etoposide (VP16), etoposide phosphate,teniposide, etc.), antitumor antibiotics (e.g., doxorubicin, adriamycin,daunorubicin, epirubicin, actinomycin, bleomycin, mitomycin,mitoxantrone, plicamycin, etc.), platinum-based compounds (e.g.cisplatin, oxaloplatin, carboplatin), anthracenedione (e.g.,mitoxantrone), substituted urea (e.g., hydroxyurea), methyl hydrazinederivative (e.g., procarbazine), or adrenocortical suppressant (e.g.,mitotane, aminoglutethimide), epipodophyllotoxins (e.g., etoposide).

Further examples of anti-cancer agents include, but are not limited to,antibiotics (e.g., daunorubicin, doxorubicin, bleomycin), enzymes (e.g.,L-asparaginase), inhibitors of mitogen-activated protein kinasesignaling (e.g. U0126, PD98059, PD184352, PD0325901, ARRY-142886,SB239063, SP600125, BAY 43-9006, wortmannin, or LY294002), mTORinhibitors, antibodies (e.g., rituxan), 5-aza-2′-deoxycytidine,doxorubicin, vincristine, etoposide, gemcitabine, imatinib(Gleevec.RTM.), geldanamycin, 17-N-Allylamino-17-Demethoxygeldanamycin(17-AAG), bortezomib, trastuzumab, anastrozole; angiogenesis inhibitors;antiandrogen, antiestrogen; antisense oligonucleotides; apoptosis genemodulators; apoptosis regulators; arginine deaminase; BCR/ABLantagonists; beta lactam derivatives; bFGF inhibitor; bicalutamide;camptothecin derivatives; casein kinase inhibitors (ICOS); clomifeneanalogues; cytarabine dacliximab; dexamethasone; estrogen agonists;estrogen antagonists; etanidazole; etoposide phosphate; exemestane;fadrozole; finasteride; fludarabine; fluorodaunorunicin hydrochloride;gadolinium texaphyrin; gallium nitrate; gelatinase inhibitors;gemcitabine; glutathione inhibitors; hepsulfam; immunostimulantpeptides; insulin-like growth factor-1 receptor inhibitor; interferonagonists; interferons; interleukins; letrozole; leukemia inhibitingfactor; leukocyte alpha interferon; leuprolide+estrogen+progesterone;leuprorelin; matrilysin inhibitors; matrix metalloproteinase inhibitors;MIF inhibitor; mifepristone; mismatched double stranded RNA; monoclonalantibody,; mycobacterial cell wall extract; nitric oxide modulators;oxaliplatin; panomifene; pentrozole; phosphatase inhibitors; plasminogenactivator inhibitor; platinum complex; platinum compounds; prednisone;proteasome inhibitors; protein A-based immune modulator; protein kinaseC inhibitor; protein kinase C inhibitors, protein tyrosine phosphataseinhibitors; purine nucleoside phosphorylase inhibitors; ras farnesylprotein transferase inhibitors; ras inhibitors; ras-GAP inhibitor;ribozymes; signal transduction inhibitors; signal transductionmodulators; single chain antigen-binding protein; stem cell inhibitor;stem-cell division inhibitors; stromelysin inhibitors; syntheticglycosaminoglycans; tamoxifen methiodide; telomerase inhibitors; thyroidstimulating hormone; translation inhibitors; tyrosine kinase inhibitors;urokinase receptor antagonists; steroids (e.g., dexamethasone),finasteride, aromatase inhibitors, gonadotropin-releasing hormoneagonists (GnRH) such as goserelin or leuprolide, adrenocorticosteroids(e.g., prednisone), progestins (e.g., hydroxyprogesterone caproate,megestrol acetate, medroxyprogesterone acetate), estrogens (e.g.,diethlystilbestrol, ethinyl estradiol), antiestrogen (e.g., tamoxifen),androgens (e.g., testosterone propionate, fluoxymesterone), antiandrogen(e.g., flutamide), immunostimulants (e.g., Bacillus Calmette-Guérin(BCG), levamisole, interleukin-2, alpha-interferon, etc.), monoclonalantibodies (e.g., anti-CD20, anti-HER2, anti-CD52, anti-HLA-DR, andanti-VEGF monoclonal antibodies), immunotoxins (e.g., anti-CD33monoclonal antibody-calicheamicin conjugate, anti-CD22 monoclonalantibody-pseudomonas exotoxin conjugate, etc.), radioimmunotherapy(e.g., anti-CD20 monoclonal antibody conjugated to ¹¹¹In ⁹⁰Y, or ¹³¹I,etc.), triptolide, homoharringtonine, dactinomycin, doxorubicin,epirubicin, topotecan, itraconazole, vindesine, cerivastatin,vincristine, deoxyadenosine, sertraline, pitavastatin, irinotecan,clofazimine, 5-nonyloxytryptamine, vemurafenib, dabrafenib, erlotinib,gefitinib, EGFR inhibitors, epidermal growth factor receptor(EGFR)-targeted therapy or therapeutic (e.g. gefitinib (Iressa™)erlotinib (Tarceva™), cetuximab (Erbitux™), lapatinib (Tykerb™),panitumumab (Vectibix™) vandetanib (Caprelsa™), afatinib/BIBW2992,CI-1033/canertinib, neratinib/HKI-272, CP-724714, TAK-285, AST-1306,ARRY334543, ARRY-380, AG-1478, dacomitinib/PF299804, OSI-420/desmethylerlotinib, AZD8931, AEE788, pelitinib/EKB-569, CUDC-101, WZ8040, WZ4002,WZ3146, AG-490, XL647, PD153035, BMS-599626), sorafenib, imatinib,sunitinib, dasatinib, or the like.

“Chemotherapeutic” or “chemotherapeutic agent” is used in accordancewith its plain ordinary meaning and refers to a chemical composition orcompound having antineoplastic properties or the ability to inhibit thegrowth or proliferation of cells.

Additionally, the nucleic acid compound described herein can beco-administered with conventional immunotherapeutic agents including,but not limited to, immunostimulants (e.g., Bacillus Calmette-Guérin(BCG), levamisole, interleukin-2, alpha-interferon, etc.), monoclonalantibodies (e.g., anti-CD2O, anti-HER2, anti-CD52, anti-HLA-DR, andanti-VEGF monoclonal antibodies), immunotoxins (e.g., anti-CD33monoclonal antibody-calicheamicin conjugate, anti-CD22 monoclonalantibody-pseudomonas exotoxin conjugate, etc.), and radioimmunotherapy(e.g., anti-CD20 monoclonal antibody conjugated to 111In, ⁹⁰Y, or ¹³¹I,etc.).

In a further embodiment, the nucleic acid compounds described herein canbe co-administered with conventional radiotherapeutic agents including,but not limited to, radionuclides such as ⁴⁷Sc, ⁶⁴Cu, ⁶⁷Cu, ⁸⁹Sr, ⁸⁶Y,⁸⁷Y, ⁹⁰Y, ¹⁰⁵Rh, ¹¹¹Ag, ¹¹¹In, ^(117m)Sn, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu,¹⁸⁶Re, ¹⁸⁸Re, ²¹¹At, and ²¹²Bi, optionally conjugated to antibodiesdirected against tumor antigens.

The term “sample” includes sections of tissues such as biopsy andautopsy samples, and frozen sections taken for histological purposes.Such samples include blood and blood fractions or products (e.g., bonemarrow, serum, plasma, platelets, red blood cells, and the like),sputum, tissue, cultured cells (e.g., primary cultures, explants, andtransformed cells), stool, urine, other biological fluids (e.g.,prostatic fluid, gastric fluid, intestinal fluid, renal fluid, lungfluid, cerebrospinal fluid, and the like), etc. A sample is typicallyobtained from a “subject” such as a eukaryotic organism, most preferablya mammal such as a primate, e.g., chimpanzee or human; cow; dog; cat; arodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; orfish. In some embodiments, the sample is obtained from a human.

A “control” sample or value refers to a sample that serves as areference, usually a known reference, for comparison to a test sample.For example, a test sample can be taken from a test condition, e.g., inthe presence of a test compound, and compared to samples from knownconditions, e.g., in the absence of the test compound (negativecontrol), or in the presence of a known compound (positive control). Acontrol can also represent an average value gathered from a number oftests or results. One of skill in the art will recognize that controlscan be designed for assessment of any number of parameters. For example,a control can be devised to compare therapeutic benefit based onpharmacological data (e.g., half-life) or therapeutic measures (e.g.,comparison of side effects). One of skill in the art will understandwhich controls are valuable in a given situation and be able to analyzedata based on comparisons to control values. Controls are also valuablefor determining the significance of data. For example, if values for agiven parameter are widely variant in controls, variation in testsamples will not be considered as significant.

“Disease” or “condition” refer to a state of being or health status of apatient or subject capable of being treated with a compound,pharmaceutical composition, or method provided herein. In embodiments,the disease is cancer (e.g. prostate cancer, renal cancer, metastaticcancer, melanoma, castration-resistant prostate cancer, breast cancer,triple negative breast cancer, glioblastoma, ovarian cancer, lungcancer, squamous cell carcinoma (e.g., head, neck, or esophagus),colorectal cancer, leukemia, acute myeloid leukemia, lymphoma, B celllymphoma, or multiple myeloma), an infectious disease (e.g., HIVinfection), an inflammatory disease (e.g., rheumatoid arthritis) or ametabolic disease (e.g., diabetes). In embodiments, the disease is adisease related to (e.g. caused by) an increase in the level of a STATtranscription factor (e.g. STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B,or STATE), STAT phosphorylation, or a STAT transcription factor or STATpathway activity, or pathway activated by a STAT transcription. In someembodiments, the disease is cancer (e.g. prostate cancer, renal cancer,metastatic cancer, melanoma, castration-resistant prostate cancer,breast cancer, triple negative breast cancer, glioblastoma, ovariancancer, lung cancer, squamous cell carcinoma (e.g., head, neck, oresophagus), colorectal cancer, leukemia, acute myeloid leukemia,lymphoma, B cell lymphoma, or multiple myeloma). In embodiments, thedisease is a viral disease (e.g. HIV infection associated disease). Inembodiments, the viral disease is associated with STAT3-dependentimmunosuppression.

As used herein, the term “cancer” refers to all types of cancer,neoplasm or malignant tumors found in mammals, including leukemia,lymphoma, carcinomas and sarcomas. Exemplary cancers that may be treatedwith a compound, pharmaceutical composition, or method provided hereininclude lymphoma, sarcoma, bladder cancer, bone cancer, brain tumor,cervical cancer, colon cancer, esophageal cancer, gastric cancer, headand neck cancer, kidney cancer, myeloma, thyroid cancer, leukemia,prostate cancer, breast cancer (e.g. triple negative, ER positive, ERnegative, chemotherapy resistant, herceptin resistant, HER2 positive,doxorubicin resistant, tamoxifen resistant, ductal carcinoma, lobularcarcinoma, primary, metastatic), ovarian cancer, pancreatic cancer,liver cancer (e.g.hepatocellular carcinoma) , lung cancer (e.g.non-small cell lung carcinoma, squamous cell lung carcinoma,adenocarcinoma, large cell lung carcinoma, small cell lung carcinoma,carcinoid, sarcoma), glioblastoma multiforme, glioma, melanoma, prostatecancer, castration-resistant prostate cancer, breast cancer, triplenegative breast cancer, glioblastoma, ovarian cancer, lung cancer,squamous cell carcinoma (e.g., head, neck, or esophagus), colorectalcancer, leukemia, acute myeloid leukemia, lymphoma, B cell lymphoma, ormultiple myeloma. Additional examples include, cancer of the thyroid,endocrine system, brain, breast, cervix, colon, head & neck, esophagus,liver, kidney, lung, non-small cell lung, melanoma, mesothelioma, ovary,sarcoma, stomach, uterus or Medulloblastoma, Hodgkin's Disease,Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, glioma,glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primarythrombocytosis, primary macroglobulinemia, primary brain tumors, cancer,malignant pancreatic insulanoma, malignant carcinoid, urinary bladdercancer, premalignant skin lesions, testicular cancer, lymphomas, thyroidcancer, neuroblastoma, esophageal cancer, genitourinary tract cancer,malignant hypercalcemia, endometrial cancer, adrenal cortical cancer,neoplasms of the endocrine or exocrine pancreas, medullary thyroidcancer, medullary thyroid carcinoma, melanoma, colorectal cancer,papillary thyroid cancer, hepatocellular carcinoma, Paget's Disease ofthe Nipple, Phyllodes Tumors, Lobular Carcinoma, Ductal Carcinoma,cancer of the pancreatic stellate cells, cancer of the hepatic stellatecells, or prostate cancer.

The term “leukemia” refers broadly to progressive, malignant diseases ofthe blood-forming organs and is generally characterized by a distortedproliferation and development of leukocytes and their precursors in theblood and bone marrow. Leukemia is generally clinically classified onthe basis of (1) the duration and character of the disease-acute orchronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid(lymphogenous), or monocytic; and (3) the increase or non-increase inthe number abnormal cells in the blood-leukemic or aleukemic(subleukemic). Exemplary leukemias that may be treated with a compound,pharmaceutical composition, or method provided herein include, forexample, acute nonlymphocytic leukemia, chronic lymphocytic leukemia,acute granulocytic leukemia, chronic granulocytic leukemia, acutepromyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, aleukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovineleukemia, chronic myelocytic leukemia, leukemia cutis, embryonalleukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia,hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia,stem cell leukemia, acute monocytic leukemia, leukopenic leukemia,lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia,lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia,mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia,monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloidgranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasmacell leukemia, multiple myeloma, plasmacytic leukemia, promyelocyticleukemia, Rieder cell leukemia, Schilling's leukemia, stem cellleukemia, subleukemic leukemia, or undifferentiated cell leukemia.

The term “sarcoma” generally refers to a tumor which is made up of asubstance like the embryonic connective tissue and is generally composedof closely packed cells embedded in a fibrillar or homogeneoussubstance. Sarcomas that may be treated with a compound, pharmaceuticalcomposition, or method provided herein include a chondrosarcoma,fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma,Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft partsarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma,chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrialsarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblasticsarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma,idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcomaof B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen'ssarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma,leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma,reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovialsarcoma, or telangiectaltic sarcoma.

The term “melanoma” is taken to mean a tumor arising from themelanocytic system of the skin and other organs. Melanomas that may betreated with a compound, pharmaceutical composition, or method providedherein include, for example, acral-lentiginous melanoma, amelanoticmelanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma,Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma,malignant melanoma, nodular melanoma, subungal melanoma, or superficialspreading melanoma.

The term “carcinoma” refers to a malignant new growth made up ofepithelial cells tending to infiltrate the surrounding tissues and giverise to metastases. Exemplary carcinomas that may be treated with acompound, pharmaceutical composition, or method provided herein include,for example, medullary thyroid carcinoma, familial medullary thyroidcarcinoma, acinar carcinoma, acinous carcinoma, adenocystic carcinoma,adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenalcortex, alveolar carcinoma, alveolar cell carcinoma, basal cellcarcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamouscell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma,bronchogenic carcinoma, cerebriform carcinoma, cholangiocellularcarcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma,corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinomacutaneum, cylindrical carcinoma, cylindrical cell carcinoma, ductcarcinoma, ductal carcinoma, carcinoma durum, embryonal carcinoma,encephaloid carcinoma, epiermoid carcinoma, carcinoma epithelialeadenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum,gelatiniforni carcinoma, gelatinous carcinoma, giant cell carcinoma,carcinoma gigantocellulare, glandular carcinoma, granulosa cellcarcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellularcarcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypernephroidcarcinoma, infantile embryonal carcinoma, carcinoma in situ,intraepidermal carcinoma, intraepithelial carcinoma, Krompecher'scarcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticularcarcinoma, carcinoma lenticulare, lipomatous carcinoma, lobularcarcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullarycarcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma,carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma,carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes,nasopharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans,osteoid carcinoma, papillary carcinoma, periportal carcinoma,preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma,renal cell carcinoma of kidney, reserve cell carcinoma, carcinomasarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinomascroti, signet-ring cell carcinoma, carcinoma simplex, small-cellcarcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cellcarcinoma, carcinoma spongiosum, squamous carcinoma, squamous cellcarcinoma, string carcinoma, carcinoma telangiectaticum, carcinomatelangiectodes, transitional cell carcinoma, carcinoma tuberosum,tubular carcinoma, tuberous carcinoma, verrucous carcinoma, or carcinomavillosum.

As used herein, the terms “metastasis,” “metastatic,” and “metastaticcancer” can be used interchangeably and refer to the spread of aproliferative disease or disorder, e.g., cancer, from one organ oranother non-adjacent organ or body part. Cancer occurs at an originatingsite, e.g., breast, which site is referred to as a primary tumor, e.g.,primary breast cancer. Some cancer cells in the primary tumor ororiginating site acquire the ability to penetrate and infiltratesurrounding normal tissue in the local area and/or the ability topenetrate the walls of the lymphatic system or vascular systemcirculating through the system to other sites and tissues in the body. Asecond clinically detectable tumor formed from cancer cells of a primarytumor is referred to as a metastatic or secondary tumor. When cancercells metastasize, the metastatic tumor and its cells are presumed to besimilar to those of the original tumor. Thus, if lung cancermetastasizes to the breast, the secondary tumor at the site of thebreast consists of abnormal lung cells and not abnormal breast cells.The secondary tumor in the breast is referred to a metastatic lungcancer. Thus, the phrase metastatic cancer refers to a disease in whicha subject has or had a primary tumor and has one or more secondarytumors. The phrases non-metastatic cancer or subjects with cancer thatis not metastatic refers to diseases in which subjects have a primarytumor but not one or more secondary tumors. For example, metastatic lungcancer refers to a disease in a subject with or with a history of aprimary lung tumor and with one or more secondary tumors at a secondlocation or multiple locations, e.g., in the breast.

As used herein, an “inflammatory disease” refers to a disease ordisorder associated with abnormal or altered inflammation. Inflammationis a biological response initiated by the immune system as part of thehealing process in response to a pathogen, damaged cells or tissues orirritants. Chronic inflammation can lead to a variety of diseases.Inflammatory diseases include, but are not limited to, atherosclerosis,allergies, asthma, rheumatoid arthritis, transplant rejection, celiacdisease, chronic prostatitis, inflammatory bowel diseases, pelvicinflammatory diseases, and inflammatory myopathies.

As used herein, “metabolic disorders” refer to diseases or disordersinvolving abnormal metabolism of a variety of molecules and substancesincluding, for example, glucose, carbohydrates, amino acids, and organicacids. Metabolic disorders include, but are not limited to, disorders ofglucose metabolism (e.g., type I and type II diabetes), disorders ofcarbohydrate metabolism, e.g., glycogen storage disease, disorders ofamino acid metabolism, e.g., phenylketonuria, maple syrup urine disease,glutaric acidemia type 1, urea cycle disorder or urea cycle defects,e.g., carbamoyl phosphate synthetase I deficiency, disorders of organicacid metabolism (organic acidurias), e.g., alcaptonuria, disorders offatty acid oxidation and mitochondrial metabolism, e.g., medium-chainacyl-coenzyme A dehydrogenase deficiency, disorders of porphyrinmetabolism, e.g., acute intermittent porphyria, disorders of purine orpyrimidine metabolism, e.g., Lesch-Nyhan syndrome, disorders of steroidmetabolism, e.g., lipoid congenital adrenal hyperplasia, congenitaladrenal hyperplasia, disorders of mitochondrial function, e.g.,Kearns-Sayre syndrome, disorders of peroxisomal function, e.g.,Zellweger syndrome, and lysosomal storage disorders, e.g., Gaucher'sdisease, and Niemann Pick disease.

As used herein, the term “infectious disease” refers to diseases ordisorders associate with infection, presence and/or growth of apathogenic agent in a host subject. Infectious pathogenic agentsinclude, but are not limited to, viruses, bacteria, fungi, protozoa,multicellular parasites and aberrant proteins, e.g., prions. Virusesassociated with infectious disease include but are not limited to,herpes simplex viruses, cytomegalovirus, Epstein-Barr virus,Varicella-zoster virus, herpes viruses, Vesicular stomatitis virus,Hepatitis viruses, Rhinovirus, Coronavirus, Influenza viruses, Measlesvirus, Polyomavirus, Human Papilomavirus, Respiratory syncytial virus,Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus,Rabies virus, Rous sarcoma virus, Yellow fever virus, Ebola virus,Simian Immunodeficiency viruses (SIV), Human Immunodeficiency viruses(HIV). Bacteria associated with infectious disease include, but are notlimited to, M. tuberculosis, Salmonella species, E. coli, Chlamydiaspecies, Staphylococcus species, Bacillus species, and Pseudomonasspecies.

The term “associated” or “associated with” in the context of a substanceor substance activity or function associated with a disease (e.g.,diabetes, cancer (e.g. prostate cancer, renal cancer, metastatic cancer,melanoma, castration-resistant prostate cancer, breast cancer, triplenegative breast cancer, glioblastoma, ovarian cancer, lung cancer,squamous cell carcinoma (e.g., head, neck, or esophagus), colorectalcancer, leukemia, acute myeloid leukemia, lymphoma, B cell lymphoma, ormultiple myeloma)) means that the disease (e.g., diabetes, cancer (e.g.prostate cancer, renal cancer, metastatic cancer, melanoma,castration-resistant prostate cancer, breast cancer, triple negativebreast cancer, glioblastoma, ovarian cancer, lung cancer, squamous cellcarcinoma (e.g., head, neck, or esophagus), colorectal cancer, leukemia,acute myeloid leukemia, lymphoma, B cell lymphoma, or multiple myeloma)or viral disease (e.g., HIV infection associated disease)) is caused by(in whole or in part), or a symptom of the disease is caused by (inwhole or in part) the substance or substance activity or function. Forexample, a symptom of a disease or condition associated with an increasein STAT3 activity may be a symptom that results (entirely or partially)from an increase in STAT3 activity (e.g., increase in STAT3transcriptional activation, increase in STAT3 activation of a signaltransduction or signaling pathway). As used herein, what is described asbeing associated with a disease, if a causative agent, could be a targetfor treatment of the disease. For example, a disease associated withincreased STAT3 activity (e.g., increase in STAT3 transcriptionalactivation, increase in STAT3 activation of a signal transduction orsignaling pathway), may be treated with an agent (e.g., compound asdescribed herein) effective for decreasing the level of activity ofSTAT3 or STAT3 pathway. For example, a disease associated with STAT3,may be treated with an agent (e.g., compound as described herein)effective for decreasing the level of activity of STAT3 or a downstreamcomponent or effector of STAT3. For example, a symptom of a disease orcondition associated with an increase in STAT (e.g., STAT1, STAT2,STAT3, STAT4, STAT5A, STAT5B, or STAT6) activity may be a symptom thatresults (entirely or partially) from an increase in STAT (e.g., STAT1,STAT2, STAT3, STAT4, STAT5A, STAT5B, or STAT6) activity (e.g., increasein STAT (e.g., STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, or STAT6)transcriptional activation, increase in STAT (e.g., STAT1, STAT2, STAT3,STAT4, STAT5A, STAT5B, or STAT6) activation of a signal transduction orsignaling pathway).

As used herein, what is described as being associated with a disease, ifa causative agent, could be a target for treatment of the disease. Forexample, a disease associated with increased STAT (e.g. STAT1, STAT2,STAT3, STAT4, STAT5A, STAT5B, or STAT6) activity (e.g., increase in STAT(e.g., STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, or STAT6)transcriptional activation, increase in STAT (e.g., STAT1, STAT2, STAT3,STAT4, STAT5A, STAT5B, or STAT6) activation of a signal transduction orsignaling pathway), may be treated with an agent (e.g., compound asdescribed herein) effective for decreasing the level of activity of aSTAT transcription factor (e.g. STAT1, STAT2, STAT3, STAT4, STAT5A,STAT5B, or STAT6) or a STAT transcription factor (e.g., STAT1, STAT2,STAT3, STAT4, STAT5A, STAT5B, or STAT6) pathway. For example, a diseaseassociated with a STAT transcription factor (e.g., STAT1, STAT2, STAT3,STAT4, STAT5A, STAT5B, or STAT6) may be treated with an agent (e.g.,compound as described herein) effective for decreasing the level ofactivity of a STAT transcription factor (e.g. STAT1, STAT2, STAT3,STAT4, STAT5A, STAT5B, or STAT6) or a downstream component or effectorof a STAT transcription factor (e.g., STAT1, STAT2, STAT3, STAT4,STAT5A, STAT5B, or STAT6).

As used herein, “treatment” or “treating,” or “palliating” or“ameliorating” are used interchangeably herein. These terms refer to anapproach for obtaining beneficial or desired results including but notlimited to therapeutic benefit and/or a prophylactic benefit. Bytherapeutic benefit is meant eradication or amelioration of theunderlying disorder being treated. Also, a therapeutic benefit isachieved with the eradication or amelioration of one or more of thephysiological symptoms associated with the underlying disorder such thatan improvement is observed in the patient, notwithstanding that thepatient may still be afflicted with the underlying disorder. Forprophylactic benefit, the compositions may be administered to a patientat risk of developing a particular disease, or to a patient reportingone or more of the physiological symptoms of a disease, even though adiagnosis of this disease may not have been made. Treatment includespreventing the disease, that is, causing the clinical symptoms of thedisease not to develop by administration of a protective compositionprior to the induction of the disease; suppressing the disease, that is,causing the clinical symptoms of the disease not to develop byadministration of a protective composition after the inductive event butprior to the clinical appearance or reappearance of the disease;inhibiting the disease, that is, arresting the development of clinicalsymptoms by administration of a protective composition after theirinitial appearance; preventing re-occurring of the disease and/orrelieving the disease, that is, causing the regression of clinicalsymptoms by administration of a protective composition after theirinitial appearance. For example, certain methods herein treat cancer(e.g. prostate cancer, renal cancer, metastatic cancer, melanoma,castration-resistant prostate cancer, breast cancer, triple negativebreast cancer, glioblastoma, ovarian cancer, lung cancer, squamous cellcarcinoma (e.g., head, neck, or esophagus), colorectal cancer, leukemia,acute myeloid leukemia, lymphoma, B cell lymphoma, or multiple myeloma).For example certain methods herein treat cancer by decreasing orreducing or preventing the occurrence, growth, metastasis, orprogression of cancer; or treat cancer by decreasing a symptom ofcancer. Symptoms of cancer (e.g. prostate cancer, renal cancer,metastatic cancer, melanoma, castration-resistant prostate cancer,breast cancer, triple negative breast cancer, glioblastoma, ovariancancer, lung cancer, squamous cell carcinoma (e.g., head, neck, oresophagus), colorectal cancer, leukemia, acute myeloid leukemia,lymphoma, B cell lymphoma, or multiple myeloma) would be known or may bedetermined by a person of ordinary skill in the art.

The term “treating” and conjugations thereof, include prevention of aninjury, pathology, condition, or disease (e.g. preventing thedevelopment of one or more symptoms of cancer (e.g. prostate cancer,renal cancer, metastatic cancer, melanoma, castration-resistant prostatecancer, breast cancer, triple negative breast cancer, glioblastoma,ovarian cancer, lung cancer, squamous cell carcinoma (e.g., head, neck,or esophagus), colorectal cancer, leukemia, acute myeloid leukemia,lymphoma, B cell lymphoma, or multiple myeloma)). For example certainmethods herein treat viral disease (e.g., HIV infection associateddisease) by decreasing or reducing or preventing the occurrence, growth,or progression of the virus infection or virus;

or treat viral disease (e.g., HIV infection associated disease) bydecreasing a symptom of viral disease (e.g., HIV infection associateddisease).

Where combination treatments are contemplated, it is not intended thatthe agents (i.e. nucleic acid compounds) described herein be limited bythe particular nature of the combination. For example, the agentsdescribed herein may be administered in combination as simple mixturesas well as chemical hybrids. An example of the latter is where the agentis covalently linked to a targeting carrier or to an activepharmaceutical. Covalent binding can be accomplished in many ways, suchas, though not limited to, the use of a commercially availablecross-linking agent.

An “effective amount” is an amount sufficient to accomplish a statedpurpose (e.g. achieve the effect for which it is administered, treat adisease, reduce enzyme activity, reduce one or more symptoms of adisease or condition, reduce viral replication in a cell). An example ofan “effective amount” is an amount sufficient to contribute to thetreatment, prevention, or reduction of a symptom or symptoms of adisease, which could also be referred to as a “therapeutically effectiveamount.” A “reduction” of a symptom or symptoms (and grammaticalequivalents of this phrase) means decreasing of the severity orfrequency of the symptom(s), or elimination of the symptom(s). A“prophylactically effective amount” of a drug is an amount of a drugthat, when administered to a subject, will have the intendedprophylactic effect, e.g., preventing or delaying the onset (orreoccurrence) of an injury, disease, pathology or condition, or reducingthe likelihood of the onset (or reoccurrence) of an injury, disease,pathology, or condition, or their symptoms. The full prophylactic effectdoes not necessarily occur by administration of one dose, and may occuronly after administration of a series of doses. Thus, a prophylacticallyeffective amount may be administered in one or more administrations. An“activity decreasing amount,” as used herein, refers to an amount ofantagonist required to decrease the activity of an enzyme or protein(e.g. STAT3) relative to the absence of the antagonist. A “functiondisrupting amount,” as used herein, refers to the amount of antagonistrequired to disrupt the function of an enzyme or protein relative to theabsence of the antagonist. Guidance can be found in the literature forappropriate dosages for given classes of pharmaceutical products. Forexample, for the given parameter, an effective amount will show anincrease or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%,75%, 80%, 90%, or at least 100%. Efficacy can also be expressed as“-fold” increase or decrease. For example, a therapeutically effectiveamount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or moreeffect over a control. The exact amounts will depend on the purpose ofthe treatment, and will be ascertainable by one skilled in the art usingknown techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms(vols. 1-3, 1992); Lloyd, The Art, Science and Technology ofPharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999);and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003,Gennaro, Ed., Lippincott, Williams & Wilkins).

“Patient” or “subject in need thereof” refers to a living organismsuffering from or prone to a disease or condition that can be treated byusing the methods provided herein. The term does not necessarilyindicate that the subject has been diagnosed with a particular disease,but typically refers to an individual under medical supervision.Non-limiting examples include humans, other mammals, bovines, rats,mice, dogs, monkeys, goat, sheep, cows, deer, and other non-mammaliananimals. In embodiments, a patient is human.

“Contacting” is used in accordance with its plain ordinary meaning andrefers to the process of allowing at least two distinct species (e.g.chemical compounds including biomolecules, or cells) to becomesufficiently proximal to react, interact or physically touch. It shouldbe appreciated, however, that the resulting reaction product can beproduced directly from a reaction between the added reagents or from anintermediate from one or more of the added reagents which can beproduced in the reaction mixture. Contacting may include allowing twospecies to react, interact, or physically touch, wherein the two speciesmay be a nucleic acid compound as described herein and a cell (e.g.,cancer cell).

“Modulate” or “modulating” refers to the suppression, enhancement orinduction of a function or condition. For example, the nucleic acidcompounds provided herein including embodiments thereof can modulatecancer by inhibition or activation of cellular gene activity (e.g.,inhibition of STAT3 activity). For example, nucleic acid compoundsincluding a STAT3 siRNA can inhibit STAT3 activity in T cell lymphomacells thereby inhibiting or reducing growth of the T cell lymphomacells.

As defined herein, the term “inhibition”, “inhibit”, “inhibiting” andthe like in reference to an siRNA or protein-inhibitor interaction meansnegatively affecting (e.g., decreasing) the activity or function of theprotein (e.g. decreasing gene transcription or translation) relative tothe activity or function of the protein in the absence of the inhibitor.In embodiments, inhibition refers to reduction of a disease or symptomsof disease (e.g., cancer). In embodiments, inhibition refers to areduction in the activity of a signal transduction pathway or signalingpathway (e.g. cell cycle). Thus, inhibition includes, at least in part,partially or totally blocking stimulation, decreasing, preventing, ordelaying activation, or inactivating, desensitizing, or down-regulatingtranscription, translation, signal transduction or enzymatic activity orthe amount of a protein (e.g. a cellular protein or a viral protein). Inembodiments, inhibition refers to inhibition of STAT3. In embodiments,inhibition refers to inhibition of FoxP3.

The terms “inhibitor,” “repressor” or “antagonist” or “downregulator”interchangeably refer to a substance that results in a detectably lowerexpression or activity level as compared to a control. The inhibitedexpression or activity can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90% or less than that in a control. In certain instances, the inhibitionis 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more incomparison to a control. An “inhibitor” is a siRNA, (e.g., shRNA, miRNA,snoRNA), compound or small molecule that inhibits cellular function(e.g., replication) e.g., by binding, partially or totally blockingstimulation, decrease, prevent, or delay activation, or inactivate,desensitize, or down-regulate signal transduction, gene expression orenzymatic activity necessary for protein activity. Inhibition asprovided herein may also include decreasing or blocking a proteinactivity (e.g., activation of STAT3) by expressing a mutant form of saidprotein thereby decreasing or blocking its activity.

The terms “agonist,” “activator,” “upregulator,” etc. refer to asubstance capable of detectably increasing the expression or activity ofa given gene or protein. The agonist can increase expression or activity10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to acontrol in the absence of the agonist. In certain instances, expressionor activity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold orhigher than the expression or activity in the absence of the agonist.

“Pharmaceutically acceptable excipient” and “pharmaceutically acceptablecarrier” refer to a substance that aids the administration of an activeagent to and absorption by a subject and can be included in thecompositions of the present invention without causing a significantadverse toxicological effect on the patient. Non-limiting examples ofpharmaceutically acceptable excipients include water, NaCl, normalsaline solutions, lactated Ringer's, normal sucrose, normal glucose,binders, fillers, disintegrants, lubricants, coatings, sweeteners,flavors, salt solutions (such as Ringer's solution), alcohols, oils,gelatins, carbohydrates such as lactose, amylose or starch, fatty acidesters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, andthe like. Such preparations can be sterilized and, if desired, mixedwith auxiliary agents such as lubricants, preservatives, stabilizers,wetting agents, emulsifiers, salts for influencing osmotic pressure,buffers, coloring, and/or aromatic substances and the like that do notdeleteriously react with the compounds of the invention. One of skill inthe art will recognize that other pharmaceutical excipients are usefulin the present invention.

The term “pharmaceutically acceptable salt” refers to salts derived froma variety of organic and inorganic counter ions well known in the artand include, by way of example only, sodium, potassium, calcium,magnesium, ammonium, tetraalkylammonium, and the like; and when themolecule contains a basic functionality, salts of organic or inorganicacids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate,maleate, oxalate and the like.

The term “preparation” is intended to include the formulation of theactive compound with encapsulating material as a carrier providing acapsule in which the active component with or without other carriers, issurrounded by a carrier, which is thus in association with it.Similarly, cachets and lozenges are included. Tablets, powders,capsules, pills, cachets, and lozenges can be used as solid dosage formssuitable for oral administration.

As used herein, the term “administering” means oral administration,administration as a suppository, topical contact, intravenous,intraperitoneal, intramuscular, intralesional, intrathecal, intranasalor subcutaneous administration, or the implantation of a slow-releasedevice, e.g., a mini-osmotic pump, to a subject. Administration is byany route, including parenteral and transmucosal (e.g., buccal,sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal).Parenteral administration includes, e.g., intravenous, intramuscular,intra-arteriole, intradermal, subcutaneous, intraperitoneal,intraventricular, and intracranial. Other modes of delivery include, butare not limited to, the use of liposomal formulations, intravenousinfusion, transdermal patches, etc. By “co-administer” it is meant thata composition described herein is administered at the same time, justprior to, or just after the administration of one or more additionaltherapies, for example cancer therapies such as chemotherapy, hormonaltherapy, radiotherapy, or immunotherapy. The compounds of the inventioncan be administered alone or can be coadministered to the patient.Coadministration is meant to include simultaneous or sequentialadministration of the compounds individually or in combination (morethan one compound). Thus, the preparations can also be combined, whendesired, with other active substances (e.g. to reduce metabolicdegradation). The compositions of the present invention can be deliveredby transdermally, by a topical route, formulated as applicator sticks,solutions, suspensions, emulsions, gels, creams, ointments, pastes,jellies, paints, powders, and aerosols.

The term “aberrant” as used herein refers to different from normal. Whenused to described enzymatic activity, aberrant refers to activity thatis greater or less than a normal control or the average of normalnon-diseased control samples. Aberrant activity may refer to an amountof activity that results in a disease, wherein returning the aberrantactivity to a normal or non-disease-associated amount (e.g. by using amethod as described herein), results in reduction of the disease or oneor more disease symptoms.

Nucleic Acid Compositions

The nucleic acid compounds provided herein including embodiments thereofare capable of modulating (i.e. activating, inhibiting) cellularactivities by modulating the activity of cellular proteins and/ornucleic acids. Non-limiting examples of cellular proteins modulated bythe nucleic acid compounds provided herein include transcription factors(e.g., STAT proteins, SMAD proteins, forkhead proteins, steroid hormonereceptors, growth hormone receptors, progesterone receptors, estrogenreceptors, androgen receptors, E26 transformation-specific (ETS)transcription factor); cytokines (e.g., interleukin-6,interleukin-6-like cytokines, pro-inflammatory cytokines); cellularreceptors (e.g., receptor tyrosine kinases (e.g., ErbB protein family orepidermal growth factor receptor (EGFR)); non-receptor tyrosine kinases(e.g., cytokine receptors); oncogenes (e.g., Src, Janus kinases (JAKS),Abl kinases, Bruton's tyrosine kinase (BTK), protein kinase B kinases(PKB/Akt kinase), protein kinase C (PKC)); and signaling molecules(e.g., phosphatases (e.g., SHP1, SHP2, T cell protein tyrosinephosphatase (TcPTP)), kinases (e.g., tyrosine kinases)).

The nucleic acid compounds provided herein include a monomeric CTLA-4aptamer nucleic acid conjugated to a cell activity modulating nucleicacid. Thus, in one aspect a nucleic acid compound including a monomericCTLA-4 aptamer nucleic acid conjugated to a cell activity modulatingnucleic acid is provided. A monomeric CTLA-4 aptamer nucleic acid asprovided herein is a nucleic acid capable of binding a CytotoxicT-Lymphocyte Antigen 4 (CTLA-4) receptor or functional fragment thereof.In embodiments, the monomeric CTLA-4 aptamer nucleic acid includes asingle stranded nucleic acid or a double stranded nucleic acid. Inembodiments, the monomeric CTLA-4 aptamer nucleic acid includes a singlestranded nucleic acid and a double stranded nucleic acid. Inembodiments, the monomeric CTLA-4 aptamer nucleic acid is a singlestranded nucleic acid. In embodiments, the monomeric CTLA-4 aptamernucleic acid is a double stranded nucleic acid. In embodiments, themonomeric CTLA-4 aptamer nucleic acid includes an RNA. In embodiments,the monomeric CTLA-4 aptamer nucleic acid includes a DNA. Inembodiments, the monomeric CTLA-4 aptamer nucleic acid is an RNA. Inembodiments, the monomeric CTLA-4 aptamer nucleic acid is a DNA. Inembodiments, the monomeric CTLA-4 aptamer nucleic acid is capable ofbinding a CTLA-4 receptor. In embodiments, the monomeric CTLA-4 aptamernucleic acid binds a CTLA-4 receptor without activating the CTLA-4receptor. In embodiments, the monomeric CTLA-4 aptamer nucleic acidspecifically binds a CTLA-4 receptor. In embodiments, the monomericCTLA-4 aptamer nucleic acid specifically binds a sub-cellularcompartment (e.g. endosome) associated CTLA-4. In embodiments, themonomeric CTLA-4 aptamer nucleic acid includes the sequence of SEQ IDNO:1. In embodiments, the monomeric CTLA-4 aptamer nucleic acid is thesequence of SEQ ID NO:1.

A monomeric CTLA-4 aptamer nucleic acid as provided herein is a nucleicacid that includes a single copy of a given aptamer unit (a monomericaptamer unit) capable of binding CTLA-4. The monomeric CTLA-4 aptamernucleic acid provided herein including embodiments thereof is notmultimeric. Thus, the monomeric CTLA-4 aptamer nucleic acid providedherein including embodiments thereof does not include a plurality of thesame aptamer unit. In embodiments, the monomeric CTLA-4 aptamer nucleicacid does not include a plurality of the same or a different aptamerunit. In embodiments, the monomeric CTLA-4 aptamer nucleic acid includesno more than one aptamer unit.

The nucleic acid compounds provided herein include a cell activitymodulating (e.g., inhibiting, activating) nucleic acid. A “cell activitymodulating nucleic acid” is a nucleic acid (e.g. DNA, RNA, polymer ofnucleotide analogs) that is capable of binding to a cellular target(e.g., nucleic acid or protein) and modulating its activity and/orfunction. A cell activity modulating nucleic acid may be a nucleic acidmodulating transcription of a target nucleic acid (e.g., mRNA from DNA)or modulating the translation of the target nucleic acid (e.g., mRNA) oraltering transcript splicing. In embodiments, the cell activitymodulating nucleic acid is a nucleic acid that is capable of binding(e.g. hybridizing) to a target nucleic acid (e.g. an mRNA translatableinto an STAT3) and modulating translation of the target nucleic acid.The target nucleic acid is or includes one or more target nucleic acidsequences to which the cell activity modulating nucleic acid binds (e.g.hybridizes). In embodiments, a cell activity modulating nucleic acid isor includes an antisense nucleic acid sequence that is capable ofhybridizing to at least a portion of a target nucleic acid at a targetnucleic acid sequence. Thus, in embodiments, the cell activitymodulating nucleic acid is an antisense nucleic acid. In embodiments,the cell activity modulating nucleic acid is a single stranded nucleicacid. In embodiments, the cell activity modulating nucleic acid is adouble stranded nucleic acid.

In embodiments, the cell activity modulating nucleic acid is aninhibitory nucleic acid. In embodiments, the inhibitory nucleic acid isan antisense nucleic acid, an RNA decoy or a ribozyme. In embodiments,the antisense nucleic acid is a siRNA. In embodiments, the cell activitymodulating nucleic acid is an antisense nucleic acid, an RNA decoy, aribozyme, a small hairpin RNA, a micro RNA or an siRNA. In embodiments,the siRNA is an anti-cancer siRNA. In embodiments, the siRNA is ananti-viral siRNA. In embodiments, the siRNA is an anti-inflammatorysiRNA. In embodiments, the siRNA is an anti-obesity siRNA. Inembodiments, the siRNA is a human siRNA. In embodiments, the siRNA is amouse siRNA. In embodiments, the siRNA is a reporter gene siRNA. Inembodiments, the reporter gene siRNA is a luciferase siRNA. Inembodiments, the siRNA is a FoxP3 siRNA. In embodiments, the siRNA is ananti-tyrosine kinase siRNA. In embodiments, the siRNA is a SignalTransducer and Activator of Transcription (STAT) siRNA. In embodiments,the STAT siRNA is a STAT3 siRNA. A STAT3 siRNA as provided herein is anantisense nucleic acid that is at least partially complementary to atleast a portion of a target nucleic acid sequence, such as an mRNAmolecule, that encodes at least a portion of the STAT3 protein.

A cell activity modulating nucleic acid as provided herein is capable ofmodulating the function and/or activity of a cellular target protein(e.g., FoxP3, STAT3). Cellular target proteins as provided hereininclude viral or bacterial proteins expressed by a cell (e.g., uponinfection). Thus, a cell activity modulating (e.g., inhibiting,activating) nucleic acid as provided herein may modulate viral andcellular proteins in a cell.

In embodiments, the cell activity modulating nucleic acid modulates atranscription factor. In embodiments, the transcription factor is a STATprotein. In embodiments, the transcription factor is a SMAD protein. Inembodiments, the transcription factor is a forkhead protein. Inembodiments, the transcription factor is a steroid hormone receptor. Inembodiments, the transcription factor is a growth hormone receptor. Inembodiments, the transcription factor is a progesterone receptor. Inembodiments, the transcription factor is an estrogen receptor. Inembodiments, the transcription factor is an androgen receptor. Inembodiments, the transcription factor is an E26 transformation-specific(ETS) transcription factor.

In embodiments, the cell activity modulating nucleic acid modulates acytokine. In embodiments, the cytokine is interleukin-6. In embodiments,the cytokine is an interleukin-6-like cytokine. In embodiments, thecytokine is a pro-inflammatory cytokine.

In embodiments, the cell activity modulating nucleic acid modulates acellular receptor. In embodiments, the cellular receptor is a receptortyrosine kinase. In embodiments, the receptor tyrosine kinase is an ErbBprotein family receptor kinase. In embodiments, the receptor tyrosinekinase is an epidermal growth factor receptor kinase. In embodiments,the cellular receptor is a non-receptor tyrosine kinase. In embodiments,the non-receptor tyrosine kinase is a cytokine receptor.

In embodiments, the cell activity modulating nucleic acid modulates anoncogene. In embodiments, the oncogene is a Src kinase. In embodiments,the oncogene is a Janus kinase. In embodiments, the oncogene is an Ablkinase. In embodiments, the oncogene is a BTK kinase. In embodiments,the oncogene is a PKB/Akt kinase. In embodiments, the oncogene is a PKCkinase.

In embodiments, the cell activity modulating nucleic acid modulates asignaling molecule. In embodiments, the signaling molecule is aphosphatase. In embodiments, the phosphatase is a SHP1 or a SHP2phosphatase. In embodiments, the phosphatase is a T cell proteintyrosine phosphatase. In embodiments, the signaling molecule is akinase. In embodiments, the kinase is a tyrosine kinase.

In embodiments, the monomeric CTLA-4 aptamer nucleic acid is conjugatedto the cell activity modulating nucleic acid through a linker. A linkeras provided herein is or includes a bond, a nucleic acid sequence,multiple nucleic acid sequences, a DNA sequence, multiple DNA sequences,an RNA sequence, multiple RNA sequences, a nucleic acid analog sequence,substituted or unsubstituted alkylene, substituted or unsubstitutedheteroalkylene, substituted or unsubstituted cycloalkylene, substitutedor unsubstituted heterocycloalkylene, substituted or unsubstitutedarylene, or substituted or unsubstituted heteroarylene.

In embodiments, the linker includes a linker nucleic acid sequence. Inembodiments, the linker nucleic acid sequence is a double-strandedlinker nucleic acid sequence. In embodiments, the linker nucleic acidsequence is a single-stranded linker nucleic acid sequence. Inembodiments, the monomeric CTLA-4 aptamer nucleic acid is connected tothe cell activity modulating nucleic acid through a linker nucleic acidsequence. Where the monomeric CTLA-4 aptamer nucleic acid is connectedto the cell activity modulating nucleic acid through a linker nucleicacid sequence, the linker nucleic acid sequence may be a single-strandedlinker nucleic acid sequence or a double-stranded linker nucleic acidsequence.

In embodiments, the monomeric CTLA-4 aptamer nucleic acid is connectedto the cell activity modulating nucleic acid through a hybridizednucleic acid overhang. Where the monomeric CTLA-4 aptamer nucleic acidis connected to the cell activity modulating nucleic acid through ahybridized nucleic acid overhang, the monomeric CTLA-4 aptamer nucleicacid includes a first single nucleic acid strand and the cell activitymodulating nucleic acid includes a second single nucleic acid strand,wherein the first nucleic acid strand includes a nucleic acid sequencethat is complementary to a nucleic acid sequence included in the secondsingle nucleic acid strand (both single nucleic acid strands includingtheir respective complementary sequences being collectively a“hybridized nucleic acid overhang”). In embodiments, the hybridizednucleic acid overhang is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 base pairslong. In embodiments, the complementary nucleic acid sequence in thehybridized nucleic acid overhang is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100base pairs long. In embodiments, the first and second single nucleicacid strands in the hybridized nucleic acid overhang are complementarythroughout their entire lengths.

In embodiments, the linker nucleic acid sequence is connected to themonomeric CTLA-4 aptamer nucleic acid through a hybridized nucleic acidoverhang. Where the linker nucleic acid sequence is connected to themonomeric CTLA-4 aptamer nucleic acid through a hybridized nucleic acidoverhang, the linker nucleic acid includes a first single nucleic acidstrand and the monomeric CTLA-4 aptamer nucleic acid includes a secondsingle nucleic acid strand, wherein the first nucleic acid strandincludes a nucleic acid sequence that is complementary to a nucleic acidsequence included in the second single nucleic acid strand (both singlenucleic acid strands including their respective complementary sequencesbeing collectively a “hybridized nucleic acid overhang”). Inembodiments, the hybridized nucleic acid overhang is 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, or 100 base pairs long. In embodiments, the complementarynucleic acid sequence in the hybridized nucleic acid overhang is 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, or 100 base pairs long. In embodiments, the firstand second single nucleic acid strands in the hybridized nucleic acidoverhang are complementary throughout their entire lengths.

In embodiments, the linker nucleic acid sequence is connected to thecell activity modulating nucleic acid through a hybridized nucleic acidoverhang. Where the linker nucleic acid sequence is connected to thecell activity modulating nucleic acid through a hybridized nucleic acidoverhang, the linker nucleic acid includes a first single nucleic acidstrand and the cell activity modulating nucleic acid includes a secondsingle nucleic acid strand, wherein the first nucleic acid strandincludes a nucleic acid sequence that is complementary to a nucleic acidsequence included in the second single nucleic acid strand (both singlenucleic acid strands including their respective complementary sequencesbeing collectively a “hybridized nucleic acid overhang”). Inembodiments, the hybridized nucleic acid overhang is 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, or 100 base pairs long. In embodiments, the complementarynucleic acid sequence in the hybridized nucleic acid overhang is 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, or 100 base pairs long. In embodiments, the firstand second single nucleic acid strands in the hybridized nucleic acidoverhang are complementary throughout their entire lengths.

In embodiments, the linker includes a non-nucleic acid spacer. Inembodiments, the monomeric CTLA-4 aptamer nucleic acid is connected tothe cell activity modulating nucleic acid through a non-nucleic acidspacer. In embodiments, the non-nucleic acid spacer is a substituted orunsubstituted alkylene, substituted or unsubstituted heteroalkylene,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene. In embodiments, thenon-nucleic acid spacer is a substituted or unsubstituted C₁-C₂₀alkylene, substituted or unsubstituted 2 to 20 membered heteroalkylene,substituted or unsubstituted C₃-C₈ cycloalkylene, substituted orunsubstituted 3 to 8 membered heterocycloalkylene, substituted orunsubstituted C₆-C₁₀ arylene, or substituted or unsubstituted 5 to 10membered heteroarylene. In embodiments, the non-nucleic acid spacer isan unsubstituted C₁-C₂₀ alkylene, unsubstituted 2 to 20 memberedheteroalkylene, unsubstituted C₃-C₈ cycloalkylene, unsubstituted 3 to 8membered heterocycloalkylene, unsubstituted C₆-C₁₀ arylene, orunsubstituted 5 to 10 membered heteroarylene. In embodiments, thenon-nucleic acid spacer is an unsubstituted C₁-C₂₀ alkylene (e.g. aC₅-C₂₀ alkylene, C₈-C₂₀ alkylene, C₁₀-C₂₀ alkylene, C₁₅-C₂₀ alkylene,C₁₈-C₂₀ alkylene or C₂₀ alkylene).

In embodiments, the linker includes a linker nucleic acid sequence and anon-nucleic acid spacer. Where the linker includes a linker nucleic acidsequence and a non-nucleic acid spacer, the monomeric CTLA-4 aptamernucleic acid may be connected to a non-nucleic acid spacer through ahybridized nucleic acid overhang and the non-nucleic acid spacer may beconnected to the cell activity modulating nucleic acid. In embodiments,the monomeric CTLA-4 aptamer nucleic acid is connected to a hybridizednucleic acid overhang through a non-nucleic acid spacer and thehybridized nucleic acid overhang is connected to the cell activitymodulating nucleic acid. In embodiments, the non-nucleic acid spacerconnects the cell activity modulating nucleic acid to the 3′ terminalend of the linker nucleic acid sequence. In embodiments, the linkerconnects the cell activity modulating nucleic acid to the 3′ terminalend of the monomeric CTLA-4 aptamer nucleic acid.

Sequences:

SEQ ID NO: 1 5′ GGG AGA GAG GAA GAG GGA UGG GCC GAC GUG CCG CA 3′SEQ ID NO: 2 (B7 binding motif of human CTLA-4):5′ QGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVID 3′SEQ ID NO: 3: (B7 binding motif of murine CTLA-4)5′ QGLRAVDTGLYLCKVELMYPPPYYVGMGNGTQIYVID 3′

Cellular Compositions

In another aspect, a mammalian cell including a nucleic acid compound asprovided herein including embodiments thereof is provided. Inembodiments, the cell expresses CTLA-4. In embodiments, the cell is amalignant cell. In embodiments, the cell is a lymphoma cell or a myelomacell. In embodiments, the cell is a lymphoma cell. In embodiments, thecell is a myeloma cell. In embodiments, the lymphoma cell is a B celllymphoma cell or a T cell lymphoma cell. In embodiments, the lymphomacell is a B cell lymphoma cell. In embodiments, the lymphoma cell is a Tcell lymphoma cell. In embodiments, the cell is an infected cell. Inembodiments, the cell is an HIV-infected cell.

In embodiments, the cell is a non-malignant cell. In embodiments, thecell is an immune cell. In embodiments, the cell is a T cell. Inembodiments, the T cell is a CD4-positive T cell, a CD8-positive T cellor a regulatory T cell. A “regulatory T cell” or “suppressor T cell” asprovided herein is a T cell which modulates the immune system, maintainstolerance to self-antigens, and abrogates autoimmune diseases.Regulatory T cells are capable of shutting down immune responses afterinvading organisms such as viruses or bacteria have been successfullyeliminated. Regulatory T cells further preventing autoimmunity byelimination auto-reactive immune cells. Non-limiting examples of surfacemolecules expressed by regulatory T cells are CD4, CD25, CTLA-4 andFoxp3.

In embodiments, the regulatory T cell is a tumor-associated regulatory Tcell. A “tumor-associated regulatory T cell” as provided herein is aregulatory T cell in a tumor environment, which after having encountereda tumor-associated (self) or tumor-specific (neo) antigen in the tumorenvironment has become activated. Under the influence of theimmunosuppressive tumor microenvironment the tumor-associated regulatoryT cell suppresses a tumor-eliminating immune response and enables tumorgrowth and expansion by promoting angiogenesis or metastasis, regulatinginflammation, and suppressing antitumor adaptive immune responses. Inembodiments, the tumor-associated regulatory T cell expresses Foxp3. Inembodiments, the tumor-associated regulatory T cell expresses CTLA-4.

In embodiments, the cell is a B cell. In embodiments, the cell is amyeloid cell. In embodiments, the cell includes a vesicle including thenucleic acid compound.

In another aspect, a cellular receptor bound to a nucleic acid compoundas provided herein including embodiments thereof is provided. Inembodiments, the cellular receptor forms part of a cell. In embodiments,the cellular receptor forms part of a vesicle within the cell. Inembodiments, the cell is a malignant cell. In embodiments, the cell is alymphoma cell or a myeloma cell. In embodiments, the cell is a lymphomacell. In embodiments, the cell is a myeloma cell. In embodiments, thelymphoma cell is a B cell lymphoma cell or a T cell lymphoma cell. Inembodiments, the lymphoma cell is a B cell lymphoma cell. Inembodiments, the lymphoma cell is a T cell lymphoma cell. Inembodiments, the cell is an infected cell. In embodiments, the cell isan HIV-infected cell.

In embodiments, the cell is a non-malignant cell. In embodiments, thecell is an immune cell. In embodiments, the cell is a T cell. Inembodiments, the T cell is a CD4-positive T cell, a CD8-positive T cellor a regulatory T cell. In embodiments, the regulatory T cell is atumor-associated regulatory T cell. In embodiments, the tumor-associatedregulatory T cell expresses Foxp3. In embodiments, the tumor-associatedregulatory T cell expresses CTLA-4. In embodiments, the cell is a Bcell. In embodiments, the cell is a myeloid cell.

Pharmaceutical Compositions

In another aspect, a pharmaceutical composition including apharmaceutically acceptable excipient and a nucleic acid compound asprovided herein including embodiments thereof (e.g., an aspect,embodiment, table, figure, claim, sequence listing or example) isprovided. In embodiments, the nucleic acid compound as provided hereinincluding embodiments thereof is included in a therapeutically effectiveamount. In embodiments, the pharmaceutical composition includes a secondtherapeutic agent. In embodiments, the second therapeutic agent is ananti-cancer agent. In embodiments, the second agent is an anti-viralagent. In embodiments, the pharmaceutical composition includes a secondagent (e.g. therapeutic agent) in a therapeutically effective amount.

Methods of Treatment

In another aspect, a method of treating a disease in a subject in needthereof is provided. The method includes administering to a subject atherapeutically effective amount of a nucleic acid compound as providedherein including embodiments thereof, thereby treating the subject.

The methods provided herein including embodiments thereof may be used totreat cancer (e.g. prostate cancer, renal cancer, metastatic cancer,melanoma, castration-resistant prostate cancer, breast cancer, triplenegative breast cancer, glioblastoma, ovarian cancer, lung cancer,squamous cell carcinoma (e.g., head, neck, or esophagus), colorectalcancer, leukemia, acute myeloid leukemia, lymphoma, B cell lymphoma, ormultiple myeloma). For example the methods provided herein includingembodiments thereof treat cancer by decreasing or reducing or preventingthe occurrence, growth, metastasis, or progression of cancer; or treatcancer by decreasing a symptom of cancer. Symptoms of cancer (e.g.prostate cancer, renal cancer, metastatic cancer, melanoma,castration-resistant prostate cancer, breast cancer, triple negativebreast cancer, glioblastoma, ovarian cancer, lung cancer, squamous cellcarcinoma (e.g., head, neck, or esophagus), colorectal cancer, leukemia,acute myeloid leukemia, lymphoma, B cell lymphoma, or multiple myeloma)would be known or may be determined by a person of ordinary skill in theart. In embodiments, the disease is cancer. In embodiments, the canceris metastatic cancer, renal cancer, colon cancer, lung cancer, melanoma,lymphoma or leukemia. In embodiments, the cancer is melanoma. Inembodiments, the cancer is lymphoma.

In embodiments, the disease is an infectious disease. In embodiments,the infectious disease is caused by HIV. In embodiments, the disease isa parasitic disease.

The methods provided herein including embodiments thereof treatinflammatory diseases (e.g. autoimmune diseases, rheumatoid arthritis,asthma, celiac diseases) by decreasing or reducing or preventing theoccurrence, growth, or progression of the inflammatory diseases (e.g.autoimmune diseases, rheumatoid arthritis, asthma, celiac diseases); ortreat inflammatory diseases (e.g. autoimmune diseases, rheumatoidarthritis, asthma, celiac diseases) by decreasing a symptom ofinflammatory diseases (e.g. autoimmune diseases, rheumatoid arthritis,asthma, celiac diseases). In embodiments, the disease is an inflammatorydisease.

The methods provided herein including embodiments thereof treatmetabolic diseases. Thus, in embodiments, the disease is a metabolicdisease. In embodiments, the disease is diabetes.

In another aspect, a method of inhibiting growth of a cancer cell isprovided. The method includes contacting a cancer cell with a nucleicacid compound as provided herein including embodiments thereof, therebyinhibiting growth of the cancer cell. In embodiments, the cancer cellincludes a level of CTLA-4 greater than a non-cancer cell. Inembodiments, the level of CTLA-4 (e.g., level of CTLA-4 protein) isabout 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 20×, 30×, 40×, 50×, 60×, 70×,80×, 90× or 100× greater than a non-cancer cell. In embodiments, thelevel of CTLA-4 (e.g., level of CTLA-4 protein) is at least about 2×,3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 20×, 30×, 40×, 50×, 60×, 70×, 80×, 90×or 100× greater than a non-cancer cell.

In embodiments, the cancer cell includes a level of STAT3 greater than anon-cancer cell. In embodiments, the level of STAT3 (e.g., level ofSTAT3 protein) is about 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 20×, 30×,40×, 50×, 60×, 70×, 80×, 90× or 100× greater than a non-cancer cell. Inembodiments, the level of STA3 (e.g., level of STAT3 protein) is atleast about 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 20×, 30×, 40×, 50×,60×, 70×, 80×, 90× or 100× greater than a non-cancer cell.

In embodiments, the method includes inducing apoptosis of the cancercell. In embodiments, the method includes inducing apoptosis in a cancercell but not in a non-cancer cell. In embodiments, the method includesinducing apoptosis in a cancer cell in a patient but not in a non-cancercell in the same patient. In embodiments, the method includes inducingapoptosis in a cancer cell but not in a non-cancer cell of the same celltype as the cancer cell (e.g. T cell, B cell, skin cell, kidney cell,lung cell, breast cell, pancreatic cell, colorectal cell, prostatecell). In embodiments, the cancer cell is in an organ. In embodiments,the cancer cell is in a lymph node. In embodiments, the cancer cell isin bone marrow.

In another aspect, a method of inhibiting activity of a tumor-associatedT cell is provided. The method includes contacting a tumor-associated Tcell with a nucleic acid compound as provided herein includingembodiments thereof, thereby inhibiting activity of the tumor-associatedT cell. In embodiments, the tumor-associated T cell includes a level ofCTLA-4 greater than a non-tumor-associated T cell. In embodiments, thelevel of CTLA-4 (e.g., level of CTLA-4 protein) is about 2×, 3×, 4×, 5×,6×, 7×, 8×, 9×, 10×, 20×, 30×, 40×, 50×, 60×, 70×, 80×, 90× or 100×greater than a non-cancer cell. In embodiments, the level of CTLA-4(e.g., level of CTLA-4 protein) is at least about 2×, 3×, 4×, 5×, 6×,7×, 8×, 9×, 10×, 20×, 30×, 40×, 50×, 60×, 70×, 80×, 90× or 100× greaterthan a non-cancer cell. In embodiments, the tumor-associated T cellincludes a level of STAT3 greater than a non-tumor-associated T cell. Inembodiments, the level of STAT3 (e.g., level of STAT3 protein) is about2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 20×, 30×, 40×, 50×, 60×, 70×, 80×,90× or 100× greater than a non-cancer cell. In embodiments, the level ofSTA3 (e.g., level of STAT3 protein) is at least about 2×, 3×, 4×, 5×,6×, 7×, 8×, 9×, 10×, 20×, 30×, 40×, 50×, 60×, 70×, 80×, 90× or 100×greater than a non-cancer cell. In embodiments, the tumor-associated Tcell expresses CD4. In embodiments, the tumor-associated T cellexpresses CD25. In embodiments, the tumor-associated T cell expressesFoxP3.

In another aspect, a method of stimulating the immune system of asubject in need thereof is provided. The method includes administeringto the subject an effective amount of a nucleic acid compound asprovided herein including embodiments thereof to a subject, therebystimulating the immune system of the subject. The stimulating mayinclude maturation, differentiation, or proliferation of natural killercells, T cells, monocytes, or macrophages. In embodiments, thestimulating includes an increase in a TH1-type immune response. Inembodiments, the stimulating includes increases in TH1-type immuneresponses. In embodiments, the method includes stimulating aplasmacytoid dendritic cell, myeloid dendritic cell, myeloid-derivedsuppressor cell, macrophage, B cell, activated NK cell, or activatedneutrophil. In embodiments, the stimulating includes increasing theactivity of a cytotoxic T cell. In embodiments, the cytotoxic T cell isa tumor-specific cytotoxic T cell.

In another aspect, a method of inhibiting a protein activity in a cellis provided. The method includes contacting a cell with a nucleic acidcompound as provided herein including embodiments thereof, therebyforming a contacted cell. The contacted cell is allowed to express theantisense nucleic acid thereby forming a cellular antisense nucleicacid. The cellular antisense nucleic acid is allowed to inhibit aprotein activity in the cell. In embodiments, the protein is a STATprotein (e.g. STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, or STATE). Inembodiments, the STAT protein is a STAT-3 protein. In embodiments, thecell is a cancer cell. In embodiments, the cell is a tumor-associated Tcell.

In another aspect, a method of modulating a protein activity in a cellis provided. The method includes contacting a cell with a nucleic acidcompound as provided herein including embodiments thereof, therebyforming a contacted cell. The contacted cell is allowed to express thecell activity modulating nucleic acid thereby forming a cellular cellactivity modulating nucleic acid. The cellular cell activity modulatingnucleic acid is allowed to modulate a protein activity in the cell. Inembodiments, the cellular cell activity modulating nucleic acidactivates a protein activity in the cell.

Disclosed are materials, compositions, and components that can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed methods and compositions. These and othermaterials are disclosed herein, and it is understood that whencombinations, subsets, interactions, groups, etc. of these materials aredisclosed that while specific reference of each various individual andcollective combinations and permutations of these compounds may not beexplicitly disclosed, each is specifically contemplated and describedherein. For example, if a method is disclosed and discussed and a numberof modifications that can be made to a number of molecules including themethod are discussed, each and every combination and permutation of themethod, and the modifications that are possible are specificallycontemplated unless specifically indicated to the contrary. Likewise,any subset or combination of these is also specifically contemplated anddisclosed. This concept applies to all aspects of this disclosureincluding, but not limited to, steps in methods using the disclosedcompositions. Thus, if there are a variety of additional steps that canbe performed, it is understood that each of these additional steps canbe performed with any specific method steps or combination of methodsteps of the disclosed methods, and that each such combination or subsetof combinations is specifically contemplated and should be considereddisclosed.

EXAMPLES

Intracellular therapeutic targets defining tumor immunosuppression inboth tumor cells and T cells remain intractable. Here, Applicants showthat covalently linking siRNA to an aptamer (apt), a RNA molecule thatselectively binds to CTLA4, allows gene silencing in exhausted CD8 Tcells and T regulatory cells in tumor, as well as CTLA4-expressingmalignant T cells. CTLA4 expression is upregulated in CD8⁺ T cells inthe tumor milieu. CTLA4^(apt)-STAT3siRNA treatments result in thechimera internalization into tumor-associated CD8⁺ T cells, silencing ofSTAT3 and activation of tumor antigen-specific T cells. Both local andsystemic administrations of CTLA4^(apt)-STA T3siRNA significantly reducetumor-associated regulatory T cells. Applicants further show thatCTLA4^(apt)-STAT3siRNA potently inhibits tumor growth and metastasis invarious mouse tumor models. Importantly, many human blood malignant Tcells express CTLA4, and CTLA4^(apt)-STA T3siRNA treatments ofimmunodeficient mice bearing human T cell lymphoma cause tumor cellapoptosis and tumor growth inhibition. Using CTLA4^(apt) as an siRNAdelivery strategy, Applicants have successfully demonstrated silencingof human and mouse STAT3 genes, as well as the luciferase gene in Tcells in vivo. Collectively, Applicants have developed a novel approachthat allows gene silencing in both tumor-associated T cells and tumorcells to inhibit tumor growth and metastasis.

CTLA4^(apt)-siRNA Uptake and Gene Silencing in T Cells

Applicants synthesized the CTLA4 targeting aptamer based on publishedsequences (Santulli-Marotto, S. et al., Cancer Res 63:7483-7489 (2003)),and chemically modified it to protect its biostability (Connolly, B. A.et al., Biochemistry 23:3443-3453 (1984); Spitzer, S. and Eckstein, F.,Nucleic Acids Res 16:11691-11704 (1988); Rettig, G. R. and Behlke, M.A., Mol Ther 20:483-512 (2011)), followed by linking it to a mouseStat3siRNA (FIG. 6A). Applicants tested primary mouse splenic cells toassess specific uptake of the CTLA4aptamer-Stat3siRNA(CTLA4^(apt)-Stat3siRNA) in immune cell populations in vitro. Eventhough CD4⁺ and CD8⁺ T cells preferentially internalizedCTLA4^(apt)-Stat3siRNA (FIG. 6B), immune cells such as macrophages anddendritic cells also took up the chimera in vivo, but to a less extent(FIG. 6C). Applicants then treated a progressive variant of fibrosarcomatumors (Dubey, P. et al., J Exp Med 185:695-705 (1997)) withCTLA4^(apt)-Stat3siRNA, to assess the silencing efficiency ofCTLA4^(apt)-Stat3siRNA in various immune subsets within the tumor. CD3⁺T cells including both CD8⁺ and CD4⁺ T cells that internalized theCTLA4^(apt)-Stat3siRNA (FITC labeled) showed significant Stat3 genesilencing in vivo (FIG. 1A). Applicants isolated CD8⁺ T cells to confirmthat CTLA4^(apt)-siRNA underwent cellular internalization and exerted agene silencing effect. Flow cytometry and live cell confocal microscopyindicated that CD8⁺ T cells internalized CTLA4^(apt)-siRNA in vitro(FIG. 1B, C), trafficking through the endosomal compartment (FIG. 1D).Real time RT-PCR and Western blotting further validated target genesilencing in these cells (FIG. 1E, F).

Although CD8⁺ T cells are known to express low levels of CTLA4, thebiological functions of CTLA4 have mainly been characterized in CD4⁺T_(Regs) and helper T cells (Wing, K. et al., Science 322:271-275(2008); Peggs, K. S. et al., J Exp Med 206:1717-1725 (2009); Pandiyan,P. et al., J Exp Med 199:831-842 (2004)). Applicants hypothesized thatCTLA4 expression might be upregulated in CD8⁺ T cells in the tumormilieu. Because IL-6 is highly expressed in many tumors including theprogressive fibrosarcoma (FIG. 7A, B), and was capable of inducing Tcell tolerance in the tumor-bearing mice (FIG. 7C-F), Applicants testedwhether IL-6 could upregulate CTLA4 expression. Western blotting showedan increase in CTLA4 protein expression (FIG. 1G), and confocalmicroscopy revealed that IL-6 treatment led to an accumulation of CTLA4protein in lipid raft domains (FIG. 1H), suggesting a functionalredistribution of CTLA4 on the surface of CD8⁺ T cells (Egen, J. G. andAllison, J. P., Immunity 16:23-35 (2002); Baroja, M. L. and Madrenas,J., Am J Transplant 3:919-926 (2003); Chikuma, S. and Bluestone, J. A.,Immunol Res 28:241-253 (2003)).

CTLA4^(apt).Stat3siRNA improves CD8⁺ T cell effector responses in vivo

Using mice bearing B16 melanoma tumors, Applicants first confirmedcellular internalization of CTLA^(apt)-Stat3siRNA in vivo by CD3⁺ Tcells and its CD8⁺ subset isolated from tumor-draining lymph nodes (FIG.2A, right panels). Notably, CTLA4^(apt)-Stat3siRNA uptake by CD8⁺ Tcells was elevated in the tumor draining lymph nodes (TDLNs), comparedwith LNs from tumor-free mice, consistent with Applicants' hypothesisthat tumor milieu/IL-6 upregulated CTLA4 expression, facilitating uptakeof the RNA chimera (FIG. 2A, middle panel). Moreover,CTLA4^(apt)-Stat3siRNA administration in vivo resulted in efficientStat3 knockdown in T cells compared to CTLA4^(apt)-LucsiRNA or vehiclecontrol treatment (FIG. 2B, C). To assess the antigen-specific CTLactivity of tumor-associated CD8⁺ T cells, Applicants adoptivelytransferred CD8^(OT-I) cells into Rag1^(−/−) mice bearing B16^(OVA)melanoma tumors. Antigen-specific production of granzyme B and IFN-γ byadoptively transferred CD8^(OT-I) cells was significantly enhanced uponCTLA4^(apt)-Stat3siRNA treatment compared with CTLA4^(apt)-LucsiRNA,vehicle control, or CD8^(OT-I) alone (FIG. 2D). Moreover,CTLA4^(apt)-Stat3siRNA treatment of B16 melanoma enhancedantigen-specific adaptive immune responses to endogenous tumor antigens,p15E and TRP-2, compared with CTLA4^(apt)-LucsiRNA, vehicle control, orCD8 T cells alone, as measured by IFN-γ production (FIG. 2E).Furthermore, CTLA4^(apt)-Stat3siRNA treatment of B16 tumors reduced PD-1expression in tumor-associated CD8⁺ T cells, in contrast toCTLA4^(apt)-LucsiRNA or vehicle control treatment (FIG. 2F), suggestingan improved CD8⁺ T cell effector population and an accumulated CTLresponse in vivo.

CTLA4^(apt.)Stat3siRNA Blocks Tumor T_(Reg) Accumulation and InhibitsTumor Growth

Since tumor-associated FoxP3⁺ T_(Regs) are a major culprit intumor-induced immunosuppresion and highly express CTLA4 (Zou),Applicants next tested the effects of CTLA4^(apt)-siRNA in targetingthis population of T cells. CTLA4^(apt)-Stat3siRNA treatment inFoxp3-GFP B16 tumor-bearing mice resulted in a substantial reduction ofFoxP3⁺ T_(Regs), shown by intravital multiphoton microscopy (FIG. 3A).Flow cytometric analysis of CD4⁺ T cells isolated from tumors of B16tumor-bearing mice confirmed a reduction in CD4⁺CD25⁺FoxP3⁺ T_(Regs)(FIG. 3B). Moreover, since IL-10 is one of the key mediators insuppression of T cell expansion by T_(Regs) and a downstream target geneof STAT3, Applicants evaluated IL-10 production by tumor-infiltratingT_(Regs). Results from this experiment showed thatCTLA4^(apt)-Stat3siRNA could effectively reduce tumor-associated T_(Reg)production of IL-10 (FIG. 3C). Applicants further tested whetherCTLA4^(apt)-Stat3siRNA could be systemically injected to achieveantitumor effects. Mice with B16 melanoma experimental lung metastaseswere treated systemically with CTLA4^(apt)-Stat3siRNA, which led to asignificant reduction of lung metastasis (FIG. 3D). Furthermore, adrastic reduction of CD4⁺Foxp3⁺ T_(Regs) (FIG. 3E) and an increase inCD8⁺ T cells in metastatic lungs were observed (FIG. 3F). In addition,CD8⁺ T cells in the lungs produced more granzyme B, supporting an activeantitumor role of CD8⁺ T cells after systemic treatment ofCTLA4^(apt)-Stat3siRNA.

The ability of CTLA4^(apt)-Stat3siRNA to silence Stat3 in both CD8⁺ andCD4⁺ T cells in the tumor suggested that CTLA4^(apt)-Stat3siRNAtreatment could induce a potent antitumor effect. In order to evaluateits therapeutic efficacy, Applicants administeredCTLA4^(apt)-Stat3siRNA, CTLA4^(apt)-LucsiRNA, or vehicle control to micebearing B16 melanoma, Renca renal cell carcinoma, A20 B cell lymphoma,or CT26 colon carcinoma tumors. Results from these experiments showedthat CTLA-4^(apt.)Stat3siRNA treatments significantly reduced tumorgrowth in all four murine tumor models (FIG. 3G).

Targeting Human CTLA4 to Deliver siRNA

The ligand binding domain of CTLA4 represented by exon 2 coding foramino acids 117-153 harbors the consensus B7 binding motif MYPPPY, whichis conserved in mouse and human (FIG. 8). This prompted Applicants toassess CTLA4 aptamer internalization in human CTLA4⁺ T cell lymphomacells. Using the CTLA4⁺ Karpas299 T cell lymphoma (FIG. 4A), Applicantsshowed that the same CTLA4 aptamers used for siRNA delivery in mousecells underwent efficient cellular internalization and co-localized withCTLA4 protein in the cytoplasm of the human T cells (FIG. 4B).Furthermore, the CTLA4 aptamer physically interacted with human CTLA4protein in a dose-dependent fashion (FIG. 4C). Of note, CTLA4 proteincomplexes of higher order (tetramers and dimers) were co-precipitated,indicating that the CTLA4 aptamer recognizes and binds to functionallyassembled CTLA4 complexes. Moreover, malignant human CTLA4⁺ T celllymphoma cells readily internalized CTLA4^(apt)-siRNA in a dose- andtime-dependent fashion as shown by flow cytometry (FIG. 4D). To assessin vivo knockdown efficiency by CTLA4^(apt)-siRNA in human T celllymphomas, Applicants treated Karpas299^(luc+) tumors in a xenograftmodel with CTLA4^(apt)-luciferase-siRNA. Compared to treatment withCTLA4 aptamer alone, the bioluminescent signal was reduced by over2-fold upon local administration of CTLA4^(apt)-luciferase-siRNA (FIG.4E), indicating a specific and robust in vivo target knockdown.

CTLA4^(apt.)STAT3siRNA Inhibits Human Lymphoma Tumor Growth

It was previously demonstrated that many types of blood malignancies,including T cell lymphomas, exhibit high CTLA4 expression (Contardi, E.et al., Int J Cancer 117:538-550 (2005)). Applicants therefore testedthe feasibility of blocking STAT3 in T cell lymphoma cells withCTLA4^(apt) linking to a human STAT3siRNA and its potential antitumoreffects. Immunohistochemical staining of human T cell lymphoma tissuesections indicated upregulated CTLA4 expression relative to normal lymphnodes (FIG. 5A, B). Treating Karpas299 human T cell lymphoma engraftedin immunodeficient mice resulted in potent tumor growth inhibition (FIG.5C). Tumor growth inhibition was associated with a drastic decrease inSTAT3 activation and induction of tumor cell apoptosis (FIG. 5D).Additionally, Applicants observed decreased proliferation, reduced tumorvasculature, and reduced B7-H1 expression (FIG. 5E).

Discussion

Given the established role of STAT3 in regulating T cell-mediated cancerprogression (Kortylewski, M. et al., Nat Med 11:1314-1321 (2005);Herrmann, A. et al., Cancer Res 70:7455-7464 (2010); Kujawski, M. etal., Cancer Res 70:9599-9610 (2010); Brayer, J. et al., Immunol Lett131:126-130 (2010)), cell-selective targeted therapeutic strategies toinhibit STAT3 activation in T cells are of tremendous interest forfuture immunotherapies. In the current study, Applicants describe anovel aptamer-based system to selectively deliver siRNA intotumor-associated T cells expressing CTLA4, including exhausted CD8 Tcells and T_(Regs), as well as malignant T cells. CTLA4^(apt)-siRNAtreatment enables silencing of intracellular checkpoints that aredifficult to target with antibodies and small-molecule drugs.CTLA4V-STAT3siRNA treatments improve endogenous adaptive effectorfunctions and induce direct tumor cell killing. While only STAT3 as atherapeutic target in CTLA4-positive cells was tested in the currentstudy, it is anticipated that the CTLA4^(apt)-siRNA conjugates areapplicable for other checkpoints and immunosuppressive molecules intumor-associated T cells and in CTLA-4-expressing malignant cells.

Using an antagonistic aptamer recognizing human CD4, Lieberman andcolleagues recently demonstrated interrupted HIV transmission anddesired RNAi-mediated knockdown of viral genes by the CD4-aptamer-siRNAchimeras (Wheeler, L. A. et al., J Clin Invest 121:2401-2412 (2011)). Inthese studies, the CD4^(apt)-siRNA conjugate targeted all CD4 T cellpopulations, the primary cellular target of HIV. By contrast, theimmunosuppressive tumor microenvironment drives CD8 T cells intoexhaustion and promotes T_(Regs), both associated with expression ofinhibitory co-receptors, including CTLA-4 and PD-1. Thus, Applicants'studies demonstrate the ability to target specific subsets of Tcells—the tumor-associated CD8 T cells and T_(Regs). While itsexpression is associated with CD8 T cell exhaustion, CTLA4 intracellularsignaling has been reported to possess a broad plasticity of cellularresponses ranging from inhibition of cytokine production and bluntingclonal expansion to T cell survival (Walunas, T. L. et al., Immunity1:405-413 (1994); Pandiyan, P. et al., J Exp Med 199:831-842 (2004);Linsley, P. S. et al., J Exp Med 176:1595-1604 (1992); Linsley, P. S. etal., Science 257:792-795 (1992); Madrenas, J. et al., J Immunol172:5948-5956 (2004)). In Applicants' prior investigations, Applicantsvalidated that STAT3 critically contributes to the inhibition ofadaptive antitumor immune responses (Kortylewski, M. et al., Nat Med11:1314-1321 (2005); Kujawski, M. et al., Cancer Res 70:9599-9610(2010)). These observations provided a previously unexplored opportunityto selectively target tumor-associated exhausted CD8 T cell populationsto restore effector functions and augment an antigen-specific CTLpopulation by directed STAT3 gene silencing.

CTLA4^(apt)-siRNA conjugates preferentially undergo cellularinternalization in CD4, and CD8 T cells. However, the conjugates arealso found in macrophages and dendritic cells to a lesser extent, whichpotentially could support the adaptive antitumor immune response throughSTAT3 knockdown in antigen-presenting cells. The uptake by antigenpresenting cells of aptamer-siRNA was also observed in the study usingthe CD4 aptamer-siRNA, which seemed to contribute to the efficacy of thechimera in vivo (Wheeler). Furthermore, CTLA4^(apt)-STAT3siRNAtreatments, administered locally or systemically, tremendously reduceCD4⁺CD25⁺FoxP3⁺ T_(Reg) populations in primary tumors as well as inmelanoma lung metastases, indicating modulation of the tumor immunologicenvironment in favor of an increased antitumor capability by CD8 Tcells. In mouse tumor models, CTLA4^(apt)-STAT3siRNA administrationshows a robust inhibition of tumor growth and metastasis. However,CTLA4-aptamer alone, reported to efficiently block CTLA4, did notimprove CTL effector function or impact T_(Reg) populations in thetumor. This is likely due to the fact that CTLA4 aptamer used by Gilboaand colleagues (Santulli-Marotto, S. et al., Cancer Res 63:7483-7489(2003)) was assembled into tetrameric forms of higher antagonisticactivity, while STAT3siRNA was synthetically fused to a monomeric CTLA4aptamer. However, due to the lethal hyperimmune phenotype of Ctla-4knockout mice (Tivol, E. A. et al., Immunity 3:541-547 (1995);Waterhouse, P. et al., Science 270:985-988 (1995)) and certain adverseevents in patients treated with CTLA-4 blocking antibodies (Wing, K. etal., Science 322:271-275 (2008); Leach, D. R. et al., Science271:1734-1736 (1996)), an aptamer with additional potent effectsantagonizing CTLA4 in the siRNA conjugate may not be necessary.

Besides tumor-associated T cell populations, malignant T cell lymphomaand other blood malignancies, also express CTLA4 (Wong, H. K. et al., JInvest Dermatol 126:212-219 (2006); Xerri, L. et al., J Pathol183:182-187 (1997); Kosmaczewska, A. et al., Leukemia 19:301-304(2005)). Many of these blood malignancies also display elevated STAT3activation (Scuto, A. et al., Cancer Res 71:3182-3188 (2011); Holtick,U. et al., Leukemia 19:936-944 (2005); Sommer, V. H. et al., Leukemia18:1288-1295 (2004); Liu, Y. et al., Blood 120:1458-1465 (2012)). Innon-malignant T cells, CTLA4 oligomerization on the cell surface readilyaccumulating in the immunological synapse is considered to depend onligand activation, and therefore represents a biologically active formof CTLA4 (Egen, J. G. and Allison, J. P., Immunity 16:23-35 (2002);Egen, J. G. et al., Nat Immunol 3:611-618 (2002); Darlington, P. J. etal., J Immunol 175:996-1004 (2005)). CTLA4 has also been reported toexist at least dimerized prior to ligation (Darlington, P. J. et al., JImmunol 175:996-1004 (2005); Linsley, P. S. et al., J Blot Chem270:15417-15424 (1995)), indicating the possibility that CTLA4oligomerizes in a ligand-independent manner in human T cell lymphoma.However, Applicants' results indicate that CTLA4^(apt)-STAT3siRNAefficiently inhibits T cell lymphoma growth concomitant withconsiderably reduced STAT3 activation. Compared to antagonisticantibodies targeting immune checkpoints, the CTLA4^(apt)-siRNA chimeraadditionally directly reduces tumor cell growth and theirimmunosuppressive impact on the T cells in the tumor microenvironment.

Methods

Mice and cell culture. Mouse care and experimental procedures with micewere performed under pathogen-free conditions in accordance withestablished institutional guidance and approved protocols from theResearch Animal Care Committees of the City of Hope. For subcutaneoustumor challenge, C57BL/6, Rag1(ko)Momj/B6.129S7, Foxp3-GFP(ki)/B6,Balb/c (The Jackson Laboratory), were injected with 10⁵ B16 melanoma orovalbumin expressing B16^(OVA), 2.5×10⁵ A20 lymphoma, colon carcinomaCT26, or renal clear cell carcininoma (Renca), or 10×10⁶ 8101fibrosarcoma regressor or progressor, respectively. For antigen specificanalyses, transgenic Ova TCR (OT-I) mice were obtained from The JacksonLaboratory. Athymic nu/nu mice (NCI Frederick) were engrafted with 10⁶Karpas299 or Karpas299^(luc+) human lymphoma cells s.c. into the flank.After tumors reached 5-7 mm in diameter, treatment with 782.5pmol/dose/mouse CTLA4-aptamer was administered every other day. Forexperimental induction of metastases by lung colonization, 5×10⁴ B16melanoma cells were injected i.v. via retro-orbital route. Mice whichreceived systemic tumor cell engraftment were treated every other daywith 782.5 pmol/dose/mouse CTLA4-aptamer administered intravenously.

Fibrosarcoma 8101 subclones (kind gift of Dr. Hans Schreiber, Universityof Chicago, Illinois) were cultured in DMEM medium (Gibco) supplementedwith 10% FBS (Sigma). Mouse melanoma B16 (kindly provided by Dr. DrewPardoll, Johns Hopkins, Balimore, Md.) and B16^(OVA) (provided by Dr. J.Mule, Moffitt Cancer Center, Tampa, Fla.), colorectal adenocarcinomaCT26 (ATCC), renal carcinoma Renca (provided by Dr. Alfred Chang,University of Michigan Medical Center, Ann Arbor, Mich.), A20 B celllymphoma (ATCC), and human Karpas299 T cell lymphoma (ATCC) were grownin RPMI1640 (Gibco) containing 10% FBS.

Adoptive T-cell transfer and ELISpot Assay. B16 or B16^(OVA) cells wereinjected s.c. into Rag 1^(−/−) mice and CD8 or CD8^(OT-I) T cells (8×10⁶to 10×10⁶) were adoptively transferred via retro-orbital route whentumors reached an average diameter of 5 mm. T cells were isolated fromspleens and lymph nodes of donor mice using negative selection (EasySep,StemCell Technologies). For antigen-specific resonses of CD8 T cells,5×10⁵ lymphocytes isolated from tumor-draining lymph nodes as well asfrom lymph nodes of naïve mice were seeded into a 96-well filtrationplate and the CD8 T cell effector response was recalled using 10 μg/mLpeptide (TRP2^(SVYDFFVWL), OVA^(SIINFEKL) were obtained from AnaSpec;p15E^(KSPWFTTL) was generated by the DNA/RNA and Protein Synthesis CoreFacility at the City of Hope) for 24 hours at 37° C. Peptide-specificgranzyme B and IFN-γ-positive spots were detected according to themanufacturer's instructions (R&D Systems, Diaclone).

Imaging. Indirect immunoflourescence has been carried out as describedprevioulsy (Herrmann, A. et al., Cancer Res 70:7455-7464 (2010))staining EEA1, CTLA-4, B7-H1 (Santa Cruz), Hoechst33342 (Sigma), lipidrafts (cholera toxin subunit B, Invitrogen), CD4, CD31 (BD Biosciences),Foxp3, granzyme B, Ki67 (abcam). Non-invasive bioluminescent imaging wasperformed according to the manufacturer's instructions using Ivis 100(Xenogen). D-Luciferin substrate was obtained from Caliper. In vivomultiphoton microscopy (IVMPM) of melanoma B16 tumors engrafted inC57BL/6 mice expressing GFP under control of the Foxp3 promoter, wasperformed while mice were anaesthetized with isoflurane/oxygen. ForIVMPM, an Ultima Multiphoton Microscopy System was used (PrairieTechnologies). For imaging GFP, the excitation wavelength was set toλ=890 nm. Band-pass filters optimized for GFP (BP λ=525/50 nm) was usedfor detection. Signals of the extracellular matrix are given by secondharmonic generation at excitation wavelength λ=890 nm and was detectedwith BP λ=460/50 nm.

Flow cytometry. Cell suspensions and tumor tissues were prepared asdescribed previously (Herrmann, A. et al., Cancer Res 70:7455-7464(2010)) and stained with different combinations of fluorophore-coupledantibodies to CD3, CD4, CD8, CD11b, CD11c, CD19, CD25, CD45, F4/80,CTLA4, phospho-Tyr705-Stat3, IL-10 (BD Biosciences). Annexin V-FITC waspurchased from BioVision. Fluorescence data were collected on Accuri (BDAccuri C6) and analyzed using FlowJo software (Tree Star).

Immunoblotting, immunoprecipitation, Cytokine Array, and ELISA. Wholecell lysates were prepared using RIPA lysis buffer containing 50 mM Tris(pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, 1 mM NaF, 15% glycerol,and 20 mM β-glycerolphosphate. A protease inhibitor cocktail was addedfresh to the lysis buffer (Mini Protease Inhibitor Cocktail, Roche).Normalized protein amounts were subjected to electrophoretic separationby SDS-PAGE, transferred onto nitrocellulose for western blotting, andsubsequently immunodetection was performed using antibodies againstCTLA4, STAT3 (Santa Cruz), and β-actin (Sigma). Forco-immunoprecipitation, anti-FITC antibody (Invitrogen) was used tolabel rProtein G agarose beads (Invitrogen), which were subsequentlyincubated for 16 h with whole cell lysates, subjected to electrophoreticprotein separation and western blot detection. For determination ofcytokine expression profiles, supernatants of fibrosarcoma 8101Re and8101Pro were collected from a 24 h cell culture. Tumor cell supernatantswere subjected to cytokine arrays and analyzed according to themanufacturer's instructions (RayBiotech). IL-6 cytokine production byfibrosarcoma 8101Re and 8101Pro was determined from a 24 h cell cultureas described above and analyzed according to the manufacturer'sinstructions (eBioscience).

Polymerase Chain Reaction. Transcript amplification was determined fromtotal RNA purified using RNeasy Kit (QIAGEN). cDNA was synthesized usingthe iScript cDNA Synthesis Kit (Bio-Rad). Real-time PCR was performed intriplicates using the Chromo4 Real-Time Detector (Bio-Rad). The murineGapdh housekeeping gene was used as an internal control to normalizetarget gene mRNA levels. Primers were obtained from SA Biosciences(mouse Stat3: PPM04643E-200, mouse 11-6: PPM03015A-200). p

What is claimed is:
 1. A nucleic acid compound comprising a monomericCTLA-4 aptamer nucleic acid conjugated to an antisense nucleic acid. 2.The nucleic acid compound of claim 1, wherein said antisense nucleicacid is an anti-cancer antisense nucleic acid.
 3. The nucleic acidcompound of claim 2, wherein said anti-cancer antisense nucleic acid isa FoxP3 antisense nucleic acid, an anti-tyrosine kinase antisensenucleic acid, or a Signal Transducer and Activator of Transcription(STAT) antisense nucleic acid.
 4. The nucleic acid compound of claim 1,wherein said monomeric CTLA-4 aptamer nucleic acid is conjugated to saidantisense nucleic acid through a linker.
 5. The nucleic acid compound ofclaim 4, wherein said linker comprises a non-nucleic acid spacer.
 6. Thenucleic acid compound of claim 5, wherein said non-nucleic acid spacerconnects said antisense nucleic acid to the 3′ terminal end of saidlinker nucleic acid sequence.
 7. The nucleic acid compound of claim 4,wherein said linker connects said antisense nucleic acid to the 3′terminal end of said monomeric CTLA-4 aptamer nucleic acid.
 8. Apharmaceutical composition comprising a pharmaceutically acceptableexcipient and the nucleic acid compound of claim
 1. 9. Thepharmaceutical composition of claim 8, further comprising an anti-cancer agent.
 10. A method of stimulating the immune system of a subjectin need thereof, said method comprising administering to a subject atherapeutically effective amount of a nucleic acid compound of claim 1,thereby stimulating the immune system of said subject.
 11. The method ofclaim 10, wherein stimulating the immune system comprises maturation,differentiation, or proliferation of natural killer cells, T cells,monocytes, or macrophages.
 12. The method of claim 10, whereinstimulating the immune system comprises an increase in a TH1-type immuneresponse.
 13. The method of claim 10, wherein stimulating the immunesystem comprises stimulating a plasmacytoid dendritic cell, myeloiddendritic cell, myeloid-derived suppressor cell, macrophage, B cell,activated NK cell, or activated neutrophil.
 14. The method of claim 10,wherein stimulating the immune system comprises increasing the activityof a cytotoxic T cell.
 15. The method of claim 14, wherein the cytotoxicT cell is a tumor-specific cytotoxic T cell.
 16. The method of claim 10,wherein said nucleic acid compound is a FoxP3 antisense nucleic acid, ananti-tyrosine kinase antisense nucleic acid, or a Signal Transducer andActivator of Transcription (STAT) antisense nucleic acid.
 17. The methodof claim 10, wherein said monomeric CTLA-4 aptamer nucleic acid isconjugated to said antisense nucleic acid through a linker.
 18. Themethod of claim 17, wherein said linker comprises a non-nucleic acidspacer.
 19. The method of claim 18, wherein said non-nucleic acid spacerconnects said antisense nucleic acid to the 3′ terminal end of saidlinker nucleic acid sequence.
 20. The method of claim 17, wherein saidlinker connects said antisense nucleic acid to the 3′ terminal end ofsaid monomeric CTLA-4 aptamer nucleic acid.